Gankyrin

ABSTRACT

Gankyrin having the amino acid sequence as set forth in SEQ ID NO: 2, or modified gankyrin comprising an amino acid sequence modified by the deletion and/or addition of one or a plurality of amino acids and/or the substitution with other amino acids in the amino acid sequence of SEQ ID NO: 2 and retaining the biological activity of gankyrin, a gene encoding it, and a method of preparing said protein and uses thereof.

This application is a divisional application of U.S. patent applicationSer. No. 09/509,775, filed Mar. 31, 2000, which is the national stageapplication filed under 35 U.S.C. §371 of International Application No.PCT/JP98/04467, filed Oct. 2, 1998, which claims priority from Japanesepatent application 9-286214, filed Oct. 3, 1997.

TECHNICAL FIELD

The present invention relates to a novel protein gankyrin, and to amethod of preparation and to uses thereof.

BACKGROUND ART

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers inthe Orient and Southern Africa. In the past 10 years, there have beensignificant advances in the diagnosis and treatment of HCC patients,with a result that the cases of surgical treatment are increasing (Arii,S. et al., Primary liver cancer in Japan, Springer-Verlag (1992)243-255; The Liver Cancer Study Group of Japan, Primary liver cancer inJapan, Springer-Verlag (1992) 445-453). Despite the marked progress,however, the survival rate remains low. One of the barriers to thelengthening of the survival period appears to be the occurrence ofintrahepatic regeneration of the cancer after its complete removal on amacroscopic level (The Liver Cancer Study Group of Japan, Ann. Surg.(1990) 211, 277-287; Belghiti, J. et al., Ann. Surg. (1991) 214,114-117).

In this connection, extensive efforts have been made to determine aprognosis judging factor that affects the intrahepatic regeneration andthe lengthening of the survival period. Up to now, the inventor of thepresent invention has analyzed the expression of several genes of HCC(Mise, M. et al., Hepatology (1996) 23, 455-464; Furutani, M. et al.,Hepatology (1996) 24, 1441-1445; Furutani, M. et al., Cancer Lett.(1997) 111, 191-197). As a result the present inventor has identifiedkan-1 (bile acid CoA: amino acid N-acyl transferase) mRNA as a novelprognosis judging factor. The expression of this factor is decreased inHCC with poor prognosis (Furutani, M. et al., Hepatology (1996) 24,1441-1445).

In addition to the above, novel molecular markers of HCC, that add tothe predicative value to conventional clinical prognostic factors suchas portal complications, α-fetoprotein (AFP) levels, tumor size, thenumber of tumors, and the like, are sought (The Liver Cancer Study Groupof Japan, Primary liver cancer in Japan, Springer-Verlag (1992) 445-453;The Liver Cancer Study Group of Japan, Ann. Surg. (1990) 211, 277-287;The Liver Cancer Study Group of Japan, Cancer (1994) 74, 2772-2780;Franco, D. et al., Gastroenterology (1990) 98, 733-738; Calvet, X. etal., Hepatology (1990) 12, 753-760).

DISCLOSURE OF THE INVENTION

In order to identify a molecular marker that is elevated in HCC, theinventor of the present invention has subtracted cDNA derived from thenon-cancerous portion of the liver from cDNA made from HCC of the samepatient. As a result, the inventors have isolated a novel gene,gankyrin, that consists of the ankyrin repeat motif alone and thatexhibits carcinogenicity in in vitro and in in vivo assay systems.

Thus, the present invention provides a novel gankyrin polypeptide, genesencoding it, a method of preparing said polypeptide, an antibody againstsaid polypeptide, and uses thereof.

In order to solve the above problems, the present invention provides apolypeptide comprising an amino acid sequence from Ala at position 14 toGly at position 226 of SEQ ID NO: 2 and having the biological activityof gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence modified by the deletion and/or addition of one or aplurality of amino acids and/or the substitution with other amino acids,in the amino acid sequence from Ala at position 14 to Gly at position226 of SEQ ID NO: 2 and retaining the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence from Met at position 1 to Gly at position 226 of SEQ IDNO: 2 and having the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence modified by the deletion and/or addition of one or aplurality of amino acids and/or the substitution with other amino acidsin the amino acid sequence from Met at position 1 to Gly at position 226in SEQ ID NO: 2 and retaining the biological activity of gankyrin.

The present invention also provides a polypeptide that is encoded by aDNA capable of hybridizing under a stringent condition to a DNA havingthe nucleotide sequence as set forth in SEQ ID NO: 1 and that has thebiological properties of gankyrin. The stringent condition as usedherein means, for example, a condition provided by 65° C. in 0.1×SSC and0.1% SDS.

The present invention also provides a signal-added polypeptide, in whicha signal sequence has been added to a polypeptide encoded by a DNA thatencodes a polypeptide comprising an amino acid sequence from Ala atposition 14 to Gly at position 226 of SEQ ID NO: 2 and having thebiological activity of gankyrin, a DNA that encodes a polypeptidecomprising an amino acid sequence modified by the deletion and/oraddition of one or a plurality of amino acids and/or the substitutionwith other amino acids in the amino acid sequence from Ala at position14 to Gly at position 226 of SEQ ID NO: 2 and retaining the biologicalactivity of gankyrin, or a DNA capable of hybridizing under a stringentcondition to a DNA that encodes a polypeptide having the nucleotidesequence as set forth in SEQ ID NO: 1 and having the biologicalproperties of gankyrin. The stringent condition as used herein means,for example, a condition provided by 65° C. in 0.1×SSC and 0.1% SDS.

The present invention also provides a polypeptide comprising an aminoacid sequence from Ala at position 14 to Met at position 231 of SEQ IDNO: 4 and having the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence modified by the deletion and/or addition of one or aplurality of amino acids and/or the substitution with other amino acidsin the amino acid sequence from Ala at position 14 to Met at position231 of SEQ ID NO: 4 and retaining the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence from Met at position 1 to Met at position 231 of SEQ IDNO: 4 and having the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence modified by the deletion and/or addition of one or aplurality of amino acids and/or the substitution with other amino acidsin the amino acid sequence from Met at position 1 to Met at position 231of SEQ ID NO: 4 and retaining the biological activity of gankyrin.

The present invention also provides a polypeptide that is encoded by aDNA capable of hybridizing under a stringent condition to a DNA havingthe nucleotide sequence as set forth in SEQ ID NO: 3 and that has thebiological properties of gankyrin. The stringent condition as usedherein means, for example, a condition provided by 65° C. in 0.1×SSC and0.1% SDS.

The present invention also provides a signal-added polypeptide, in whicha signal sequence has been added to a polypeptide encoded by a DNA thatencodes a polypeptide comprising an amino acid sequence from Ala atposition 14 to Met at position 231 of SEQ ID NO: 4 and having thebiological activity of gankyrin, a DNA that encodes a polypeptidecomprising an amino acid sequence modified by the deletion and/oraddition of one or a plurality of amino acids and/or the substitutionwith other amino acids in the amino acid sequence from Ala at position14 to Met at position 231 of SEQ ID NO: 4 and retaining the biologicalactivity of gankyrin, or a DNA capable of hybridizing under a stringentcondition to a DNA that encodes a polypeptide having the base sequenceas set forth in SEQ ID NO: 3 and having the biological properties ofgankyrin. The stringent condition as used herein means, for example, acondition provided by 65° C. in 0.1×SSC and 0.1% SDS.

The present invention also provides a polypeptide comprising an aminoacid sequence from Ala at position 14 to Met at position 231 of SEQ IDNO: 6 and having the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence modified by the deletion and/or addition of one or aplurality of amino acids and/or the substitution with other amino acidsin the amino acid sequence from Ala at position 14 to Met at position231 of SEQ ID NO: 6 and retaining the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence from Met at position 1 to Met at position 231 of SEQ IDNO: 6 and having the biological activity of gankyrin.

The present invention also provides a polypeptide comprising an aminoacid sequence modified by the deletion and/or addition of one or aplurality of amino acids and/or the substitution with other amino acidsin the amino acid sequence from Met at position 1 to Met at position 231of SEQ ID NO: 6 and retaining the biological activity of gankyrin.

The present invention also provides a polypeptide that is encoded by aDNA capable of hybridizing under a stringent condition to a DNA havingthe nucleotide sequence as set forth in SEQ ID NO: 5 and that has thebiological properties of gankyrin. The stringent condition as usedherein means, for example, a condition provided by 65° C. in 0.1×SSC and0.1% SDS.

The present invention also provides a signal-added polypeptide, in whicha signal sequence has been added to a polypeptide encoded by a DNA thatencodes a polypeptide comprising an amino acid sequence from Ala atposition 14 to Met at position 231 of SEQ ID NO: 6 and having thebiological activity of gankyrin, a DNA that encodes a polypeptidecomprising an amino acid sequence modified by the deletion and/oraddition of one or a plurality of amino acids and/or the substitutionwith other amino acids in the amino acid sequence from Ala at position14 to Met at position 231 of SEQ ID NO: 6 and retaining the biologicalactivity of gankyrin, or a DNA capable of hybridizing under a stringentcondition to a DNA that encodes a polypeptide having the base sequenceas set forth in SEQ ID NO: 5 and having the biological properties ofgankyrin. The stringent condition as used herein means, for example, acondition provided by 65° C. in 0.1×SSC and 0.1% SDS.

The present invention also provides a fusion polypeptide comprising theabove polypeptide and another peptide or polypeptide.

The present invention also provides a DNA encoding the abovepolypeptide.

The present invention also provides a vector comprising the above DNA.

The present invention also provides a host transformed with the abovevector.

The present invention also provides a method of preparing the abovepolypeptide, said method comprising culturing a host transformed with anexpression vector comprising a DNA encoding said polypeptide andrecovering the desired polypeptide from said culture.

The present invention also provides an antibody that specifically reactsto the above polypeptide. The antibody is preferably a monoclonalantibody or a polyclonal antibody.

The present invention also provides a method of detecting or determininga gankyrin polypeptide, said method comprising contacting the aboveantibody to a sample expected to contain said gankyrin polypeptide anddetecting or determining the formation of an immune complex between saidantibody and said gankyrin polypeptide.

The present invention also provides an antisense oligonucleotide thathybridizes any of the sites of the nucleotide sequence as set forth inSEQ ID NO: 1.

The present invention also provides an antisense oligonucleotide to atleast 20 contiguous nucleotides in the nucleotide sequence as set forthin SEQ ID NO: 1. Said antisense oligonucleotide to the at least 20contiguous nucleotides preferably have a translation initiation codon.

The present invention also provides an expression inhibitor of agankyrin polypeptide, said inhibitor comprising said antisenseoligonucleotide as an active ingredient.

The present invention also provides a method of screening an agonist oran antagonist of the gankyrin polypeptide to the binding of the gankyrinpolypeptide and Rb, said method comprising contacting a gankyrinpolypeptide or a substance containing the gankyrin polypeptide with asample expected to contain the agonist or the antagonist of the gankyrinpolypeptide in the presence of Rb, and detecting a free gankyrinpolypeptide or Rb. The above substance containing the gankyrinpolypeptide is for example a cell lysate that expresses gankyrin.

The present invention also provides a method of screening an agonist oran antagonist of the gankyrin polypeptide to the binding of the gankyrinpolypeptide and NFκB, said method comprising contacting a gankyrinpolypeptide or a material containing the gankyrin polypeptide with asample expected to contain the agonist or the antagonist of the gankyrinpolypeptide in the presence of NFκB, and detecting a free gankyrinpolypeptide or NFκB. The above substance containing the gankyrinpolypeptide is for example a cell lysate that expresses gankyrin.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a diagram showing the position of a gankyrin gene on the humanX chromosome.

FIG. 2 is a photograph showing that a gankyrin gene on the chromosome inhuman lymphocytes was fluorescence-stained by the in situ hybridizationmethod and detected.

FIG. 3 is an electrophoregram showing the result that mRNAs from thenormal liver tissue (N) and the hepatic cancer tissue (T) of 5 patientswith hepatic cancer (1 to 5) were detected by the Northern method usinghuman gankyrin cDNA as a probe.

FIG. 4 is an electrophoregram showing the result that mRNAs from varioushuman cell lines were detected by the Northern method using humangankyrin cDNA as a probe.

FIG. 5 is an electrophoregram showing the result that mRNAs from variousnormal tissues were detected by the Northern method using human gankyrincDNA as a probe.

FIG. 6 is an electrophoregram showing the result that gankyrinpolypeptides in cell lysates from the normal tissue (N) and the hepaticcancer tissue (T) of 3 patients with hepatic cancer (1 to 3) weredetected by the Western blot method using anti-gankyrin polypeptideantibody.

FIG. 7 is an electrophoregram showing the result that gankyrinpolypeptides in cell lysates from various human cell lines were detectedby the Western blot method using anti-gankyrin polypeptide antibody.

FIG. 8A is an electrophoregram showing the result that the in vitrotranslated gankyrin gene products were detected in a similar manner toFIG. 6, and FIG. 8B is an electrophoregram showing the result that thein vitro translated gankyrin gene products (non-labeled) were detectedin a similar manner to FIG. 6.

FIG. 9 is an electrophoregram showing the result that fusionpolypeptides comprising gankyrin polypeptide and GST were expressed inEscherichia coli (E. coli) and then were detected using variousantibodies.

FIG. 10 is an electrophoregram showing the result that fusionpolypeptides of gankyrin polypeptides and HA were expressed in the 293cells, immunoprecipitated and then were detected using variousantibodies.

FIG. 11 is an electrophoregram showing the result that fusionpolypeptides of gankyrin polypeptides and HA were expressed in the 293cells, immunoprecipitated and then were detected, using variousantibodies.

FIG. 12 is an electrophoregram showing the result that mRNAs in variouscell cycles of the NIH/3T3 cells were detected by the Northern methodusing mouse gankyrin cDNA as a probe.

FIG. 13 is an electrophoregram showing the result that mRNAs in theNIH/3T3 cells propagated at various concentrations were detected in asimilar manner to FIG. 12.

FIG. 14 is an electrophoregram showing the result that mRNAs in thehepatic tissue during the process of liver regeneration after partialhepatic resection in mice were detected in a similar manner to FIG. 12.

FIG. 15 is a drawing that shows the position and the number ofrepetition of ankyrin repeats in various proteins.

EMBODIMENT FOR CARRYING OUT THE INVENTION

In accordance with the present invention, a gankyrin polypeptide means apolypeptide having the biological activity of gankyrin. The biologicalactivity of gankyrin is carcinogenicity, of which specific effectsinclude, as described in Example 4, the elevation of colony-formingability of cells in soft agar and the induction of tumorigenicity andthe suppression of apoptosis in mice.

The gankyrin gene of the present invention and the cDNA thereof can beobtained by screening hepatic cancer cells or hepatic cancer tissue asits source to obtain the gene or the cDNA thereof. As a method ofscreening or isolating the gene or cDNA thereof, there can be used onethat can selectively screen the gene whose amount expressed varies, suchas the subtraction method (Nucleic Acids Research (1988) 16, 10937) andthe differential hybridization method (Cell (1979) 16, 443-452).

The gene encoding the gankyrin polypeptide of the present invention canbe obtained by the subtraction between a cDNA library prepared from thenormal liver tissue and a cDNA library prepared from the hepatic cancertissue to select cDNA derived from mRNA that is expressed in the hepaticcancer tissue but not in the normal liver tissue.

For example, in the subtraction method, cDNA obtained from the hepaticcancer tissue or the normal liver tissue is amplified. Primers toamplify the normal liver tissue are labeled with a labeling compound,for example biotin. Then, an excessive amount of double stranded cDNAderived from the normal liver tissue is mixed with a small amount ofdouble stranded cDNA derived from the hepatic cancer tissue to form amixture, which is then made single-stranded by heat denaturation, andthen returned to double strands. Among the cDNA derived from hepaticcancer tissue, most of those that were also present in the normal livertissue come to form double strands with the cDNA derived from the normalliver tissue and to be labeled.

However, when cDNAs derived from the hepatic cancer tissue form doublestrands with each other, they are not labeled. Accordingly, by removingcDNA double stranded DNA having a label, cDNA that is specific for thehepatic cancer tissue can be obtained. By repeating this procedure,cDNAs specific for the hepatic cancer tissue can be concentrated. Thespecific procedure is shown in Example 1. By using cDNA fragments orfull-length cDNAs obtained as probes, it is also possible to conductNorthern blotting on mRNA from cells or tissues that express gankyrinpolypeptides or from cells or tissues that do not express gankyrinpolypeptides and thereby to confirm that the selected gene specificallyexpresses mRNA.

By screening cDNA libraries using cDNA or cDNA fragments obtained asabove, it is possible to obtain gankyrin genes from different cells,tissues, organs or species. Furthermore, by determining the nucleotidesequence of the cDNA obtained, it is possible to determine thetranslation region that encodes a gankyrin gene product, a polypeptide,and thereby to obtain the amino acid sequence of this polypeptide. It isalso possible to isolate chromosomal DNA by screening genomic DNAlibraries using the obtained cDNA as a probe.

DNA libraries such as cDNA libraries or genomic DNA libraries may beprepared by a method described, for example, in Sambrook, J. et al.,Molecular Cloning, Cold Spring Harbor Laboratory Press (1989), orcommercially available DNA libraries may be used.

The gene or DNA of the present invention can be obtained by the PCRmethod using as a primer the nucleotide sequence or part thereof, if itis known.

The gankyrin polypeptide of the present invention includes a polypeptidethat is encoded by a DNA that hybridizes to a nucleic acid having thenucleotide sequence as set forth in SEQ ID NO: 1 under a stringentcondition, and that has the biological activity of gankyrin.

Such stringent conditions include, for example, a low stringentcondition. By way of example, a low stringent condition is 50° C. in2×SSC and 0.1% SDS. More preferably, there may be mentioned a highstringent condition. By way of example, a high stringent condition is65° C. in 0.1×SSC and 0.1% SDS.

The above hybridizing DNA is preferably naturally occurring DNA, and,for example, it may be cDNA or genomic DNA. Homology search carried outon the amino acid sequence as set forth in SEQ ID NO: 2 and thenucleotide sequence as set forth in SEQ ID NO: 1 using all the sequencescontained in the known DNA databases (GenBank, EMBL) and the proteindatabase (SWISS-PLOT) did not give any matches. From this result, it wasrevealed that the gene and the gene product polypeptide of the presentinvention are novel molecules.

As shown in Example 1, it was found that the gene that hybridizes to thecDNA of the novel gankyrin polypeptide of the present invention iswidespread in non-human animals such as rats, mice, and the like, andalso in various tissues. Thus, the above naturally occurring DNA may becDNA or genomic DNA derived from the tissues in which mRNA thathybridizes to cDNA of human gankyrin polypeptide in, for example,Example 1 is detected.

The present invention also encompasses a DNA that hybridizes to anucleic acid having the nucleotide sequence as set forth in SEQ ID NO: 2and that encodes a polypeptide having the activity of gankyrin. This DNAcan also be expressed by the above-mentioned method. In order to obtainsuch a gankyrin polypeptide, synthetic oligonucleotide primers can beused to introduce the desired mutation in the nucleotide sequence of agankyrin gene (Mark, D. F. et al., Proc. Natl. Acad. Sci. U.S.A. (1984)81, 5662-5666; Zoller, M. J. & Smith, M. Nucleic Acids Research (1982)10, 6487-6500; Wang, A. et al., Science 224, 1431-1433;Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79,6409-6413).

In addition to being cDNA and genomic DNA that encodes the gankyrinpolypeptide, it may be a synthetic DNA. Specifically, there may bementioned a DNA that encodes gankyrin having the amino acid sequence asset forth in SEQ ID NO: 2, and a DNA having the nucleotide sequence asset forth in SEQ ID NO: 1 is used. These DNAs may be produced using geneengineering technology that is known per se.

An obtained transformant Escherichia coli containing the plasmidpBS-t4-11 described hereinbelow in Example 1 was designated asEscherichia coli DH5α (pBS-t4-11) and was internationally deposited onSep. 29, 1997, with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, MITI(Higashi 1-Chome 1-3, Tsukuba-shi, Ibaraki, Japan) under the accessionnumbers FERM BP-6128.

As the gankyrin polypeptide of the present invention, there can bementioned a gankyrin polypeptide having an amino acid sequence that isidentical or substantially identical to the amino acid sequence as setforth in SEQ ID NO: 2. Specifically, in addition to gankyrin having theamino acid sequence as set forth in SEQ ID NO: 2, there can be mentionedthose in which one or more, preferably 2 or more and 30 or less, morepreferably 2 or more and 10 or less amino acids are deleted, one ormore, preferably 2 or more and 30 or less, more preferably 2 or more and10 or less amino acids are added to the amino acid sequence as set forthin SEQ ID NO: 2; or one or more, preferably 2 or more and 30 or less,more preferably 2 or more and 10 or less amino acids in the amino acidsequence as set forth in SEQ ID NO: 2 are substituted with other aminoacids.

The present invention also includes a polypeptide that has thebiological activity of gankyrin and that is homologous to a polypeptidehaving the amino acid sequence as set forth in SEQ ID NO: 2. As usedherein, “homologous polypeptides” refers to those polypeptides that havean amino acid homology of at least 70%, preferably at least 80%, morepreferably at least 90%, and more preferably at least 95% or more,generally, for at least 20, preferably 30 contiguous amino acid residuesto the amino acid sequence as set forth in SEQ ID NO: 2.

The gankyrin polypeptides of the present invention differ in amino acidsequence, molecular weight, isoelectric point, or presence or form ofsugar chains, depending on the cells or host that produce thepolypeptides, or methods of purification described hereinbelow. However,the gankyrin polypeptides obtained are included in the present inventionas long as they have the activity substantially equivalent to that of anaturally occurring gankyrin polypeptide. As the activity that issubstantially equivalent to the gankyrin polypeptide as used herein,there can be mentioned carcinogenicity as in Example 4 described below,such as the elevation of colony-forming ability of cells in soft agarand the suppression of tumorigenicity and of apoptosis induction inmice. Substantially equivalent as used herein means that carcinogenicityis equivalent in property.

As a partial peptide of the gankyrin polypeptide of the presentinvention, there can be mentioned, for example, a partial peptidecomprising one or more than one region of the hydrophobic region or thehydrophilic region estimated from the hydrophobic plot analysis amongthe gankyrin molecules. These partial peptides can include part or allof a hydrophobic region or part or all of a hydrophilic region.

The partial peptide of the gankyrin polypeptide of the present inventioncan be produced according to the peptide synthesis method that is knownper se or by cleaving the gankyrin polypeptide of the present inventionwith a suitable peptidase. The peptide synthesis method may be, forexample, a solid phase synthesis or a liquid phase synthesis.

After the reaction, the partial peptide of the present invention can beisolated and purified by combining conventional purification methodssuch as solvent extraction, distilation, column chromatography, highperformance liquid chromatography, and recrystalization.

The DNA constructed as described above can be expressed by a knownmethod to obtain a gankyrin polypeptide. When mammalian cells are used,expression may be accomplished using an expression vector containing acommonly used useful promoter/enhancer, the gene to be expressed, andDNA in which the poly A signal has been operably linked at 3′ downstreamthereof or a vector containing said DNA. Examples of thepromoter/enhancer include human cytomegalovirus immediate earlypromoter/enhancer.

Additionally, as the promoter/enhancer which can be used for expressionof gankyrin polypeptide, there can be used viral promoters/enhancerssuch as retrovirus, polyoma virus, adenovirus, and simian virus 40(SV40), and promoters/enhancers derived from mammalian cells such ashuman elongation factor 1α (HEF1α).

For example, expression may be readily accomplished by the method ofMulligan et al. (Nature (1979) 277, 108) when the SV40 promoter/enhanceris used, or by the method of Mizushima et al. (Nucleic Acids Res. (1990)18, 5322) when the HEF1α promoter/enhancer is used.

In the case of Escherichia coli (E. coli), expression may be effected byoperably linking a conventionally used useful promoter, a signalsequence for antibody secretion, and the antibody gene to be expressed,followed by expression thereof. As the promoter, for example, there canbe mentioned the lacZ promoter and the araB promoter. The method of Wardet al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427) may beused when the lacZ promoter is used, and the method of Better et al.(Science (1988) 240, 1041-1043) may be used when the araB promoter isused.

As the signal sequence for gankyrin polypeptide secretion, when producedin the periplasm of E. coli, the pelB signal sequence (Lei, S. P. etal., J. Bacteriol. (1987) 169, 4379) can be used.

As the origin of replication, there can be used those derived from SV40,polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like.Furthermore, for the amplification of gene copy number in the host cellsystem, expression vectors can include as selectable markers theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, thedihydrofolate reductase (dhfr) gene and the like.

For the production of a gankyrin polypeptide, any production system canbe used. The production system of gankyrin polypeptide preparationcomprises the in vitro or the in vivo production system. As the in vitroproduction system, there can be mentioned a production system whichemploys eukaryotic cells and the production system which employsprokaryotic cells.

When the eukaryotic cells are used, there are the production systemswhich employ animal cells, plant cells, and fungal cells. Known animalcells include (1) mammalian cells such as CHO cells (J. Exp. Med. (1995)108, 945), COS cells, myeloma cells, baby hamster kidney (BHK) cells,HeLa cells, and Vero cells, (2) amphibian cells such as Xenopus oosytes(Valle, et al., Nature (1981) 291, 358-340), or (3) insect cells such assf9, sf21, and Tn5. As CHO cells, preferably dhfr-CHO (Proc. Natl. Acad.Sci. U.S.A. (1980) 77, 4216-4220) that lacks the DHFR gene and CHO K-1(Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275) may be used.

Known plant cells include, for example, those derived from Nicotianatabacum, which is subjected to callus culture. Known fungal cellsinclude yeasts such as the genus Saccharomyces, for exampleSaccharomyces cereviceae, or filamentous fungi such as the genusAspergillus, for example Aspergillus niger.

When the prokaryotic cells are used, there are the production systemswhich employ bacterial cells. Known bacterial cells include Escherichiacoli (E. coli), and Bacillus subtilis.

By transforming these cells with the desired DNA and culturing thetransformed cells in vitro, the gankyrin polypeptide can be obtained.Culturing is carried out in a known method. For example, as the cultureliquid, DMEM, MEM, RPMI1640, and IMDM can be used, and serum supplementssuch as fetal calf serum (FCS) may be used in combination, or serum-freeculture medium may be used. pH during the culture is preferably about 6to 8. The culturing is usually conducted at about 30 to 40° C. for about15 to 200 hours with optional medium exchange, aeration and agitation.

As in vivo production systems, there can be mentioned those which employanimals and those which employ plants. The desired DNA is introducedinto an animal or a plant, and the gankyrin polypeptide is produced insuch an animal or a plant and then collected. As used herein “host”encompasses these animals and plants.

When animals are used, there are the production systems which employmammals and insects.

As mammals, goats, pigs, sheep, mice, and cattle can be used (VickiGlaser, SPECTRUM Biotechnology Applications, 1993). When mammals areused, transgenic animals can also be used.

For example, an desired DNA is inserted into the middle of the geneencoding protein which is inherently produced in the milk such as goat βcasein to prepare fusion genes. DNA fragments containing the fusion geneinto which said DNA has been inserted are injected into a goat embryo,and the embryo is introduced into a female goat. The gankyrinpolypeptide is obtained from the milk produced by the transgenic goatborn to the goat who received the embryo or offsprings thereof. In orderto increase the amount of milk containing the gankyrin polypeptideproduced by the transgenic goat, hormones may be given to the transgenicgoat as appropriate (Ebert, K. M. et al., Bio/Technology (1994) 12,699-702).

When insects are used, silkworms, for example, can be used. Whensilkworms are used, baculovirus into which the desired DNA has beeninserted is infected to the silkworm, and the desired gankyrinpolypeptide can be obtained from the body fluid of the silkworm (Susumu,M. et al., Nature (1985) 315, 592-594).

When plants are used, tabacco for example can be used. Moreover, whentabacco is used, the desired DNA is inserted into an expression vectorfor plants, for example PMON 530, and then the vector is introduced intoa bacterium such as Agrobacterium tumefaciens. The bacterium is theninfected to tabacco such as Nicotiana tabacum to obtain the desiredpolypeptide from the leaves of the tabacco (Julian, K.-C. Ma et al.,Eur. J. Immunol. (1994) 24, 131-138). As methods of introducing anexpression vector into a host, there can be used a known method such asthe calcium phosphate method (Virolgoy (1973) 52, 456-467), theelectroporation method (EMBO J. (1982) 1, 841-845), and the like.Considering the frequency of use of the host's codon for use inexpression, a sequence having a better efficiency of expression can bedesigned (Grantham, R. et al., Nucleic Acids Research (1981) 9,r43-r74).

That the gankyrin gene products thus obtained have the biologicalactivity of gankyrin can be confirmed, for example, in the followingmanner. Using, for example, a method described in Example 4 below, cellsthat produce a gankyrin polypeptide are cultured in soft agar. Gankyrinpolypeptide-expressing cells have an elevated colony-forming ability insoft agar. Alternatively, cells that express a gankyrin polypeptide aregrafted to mice. Cells that express a gankyrin polypeptide show anelevated tumorigenicity. Alternatively, cells that express a gankyrinpolypeptide are placed under a apoptosis-inducing condition. Cells thatexpress a gankyrin polypeptide suppress the induction of apoptosis.

Polypeptides obtained as described above can be isolated from the insideor outside of the host as a substantially pure homogeneous polypeptide.Separation and purification of the gankyrin polypeptide may beaccomplished by, but this is not limited to, the separation and thepurification methods conventionally used for protein purification. Forexample, the gankyrin polypeptide can be separated and purified byselecting and combining, as appropriate, methods including, but notlimited to, chromatography columns, filtration, ultrafiltration,salting-out, solvent precipitation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis,and the like.

As chromatography, there may be mentioned, for example, affinitychromatography, ion exchange chromatography, hydrophobic chromatography,gel-filtration, reverse phase chromatography, adsorption chromatography,and the like (Strategies for Protein Purification and Characterization:A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1986). These chromatographies can be carriedout using a liquid chromatography such as HPLC and FPLC. Before or afterpurification, gankyrin polypeptides may be modified by the action of asuitable protein-modifying enzyme, or peptides can partially be removed.As the protein-modifying enzymes, there may be used trypsin,chymotrypsin, lysil endopeptidase, protein kinase, glucosidase, andprotein kinase, glucosidase.

The gankyrin polypeptide of the present invention is important since itis used in a screening method. Thus, it is important since it is used ina method of screening an agonist or an antagonist of the gankyrinpolypeptide, said method comprising contacting a gankyrin polypeptide ora material containing the gankyrin polypeptide with a sample expected tocontain an agonist or an antagonist of the gankyrin polypeptide in thepresence of Rb, and detecting or determining a free gankyrin polypeptideor Rb; or a method of screening an agonist or an antagonist of thegankyrin polypeptide, said method comprising contacting a gankyrinpolypeptide or a material containing the gankyrin polypeptide with asample expected to contain the agonist or the antagonist of the gankyrinpolypeptide in the presence of NFκB (Baeuerle, P. A. et al., Cell (1988)53, 211-217), and detecting or determining a free gankyrin polypeptideor NFκB.

The gankyrin polypeptide for use in these screening methods may beeither a recombinant type or a natural type. It may also be a partialpeptide of the gankyrin polypeptide as long as it retains the propertyof binding to Rb or NFκB. As the substance containing the gankyrinpolypeptide, there may be mentioned the lysates of the cells thatexpress gankyrin polypeptides.

Thus, the present invention relates to a method of screening an agonistor an antagonist of the gankyrin polypeptide comprising comparing a freegankyrin polypeptide or Rb detected or determined when a gankyrinpolypeptide or a material containing the gankyrin polypeptide and asample expected to contain the agonist or the antagonist of the gankyrinpolypeptide are contacted, and a free gankyrin polypeptide or Rbdetected or determined when a gankyrin polypeptide or a materialcontaining the gankyrin polypeptide and a sample that does not containthe agonist or the antagonist of the gankyrin polypeptide are contacted,both in the presence of Rb.

The present invention also relates to a method of screening an agonistor an antagonist of the gankyrin polypeptide comprising comparing a freegankyrin polypeptide or NFκB detected or determined when a gankyrinpolypeptide or a material containing the gankyrin polypeptide and asample expected to contain the agonist or the antagonist of the gankyrinpolypeptide are contacted, and a free gankyrin polypeptide or NFκBdeleted or determined when a gankyrin polypeptide or a substancecontaining the gankyrin polypeptide and a sample that does not containthe agonist or the antagonist of the gankyrin polypeptide are contacted,both in the presence of NFκB.

In order to detect or determine free gankyrin polypeptides, Rb or NFκBin these screening methods, gankyrin polypeptides, Rb or NFκB arelabeled with, for example, biotin, avidin, a radioisotope such as[¹²⁵I], [³⁵S], [³H], [¹⁴C], a fluorescent substance, an enzyme such ashorseradish peroxidase and alkaline phosphatase, and then the label isdetected or determined. These labeling compounds are known and can belabeled by conventional methods. Free gankyrin polypeptides, Rb, or NFκBcan also be detected or determined using antibodies to gankyrinpolypeptides, Rb, or NFκB.

Specifically, a gankyrin polypeptide is bound to a support such as beadsor a plate, to which a sample expected to contain an agonist or anantagonist of the gankyrin polypeptide is added in the presence of Rb orNFκB, and after incubation, Rb or NFκB contained in the solution may bedetected or determined with an antibody. Alternatively, in order todetect or determine free RB or NFκB, Rb or NFκB that is bound to thegankyrin polypeptide immobilized on the plate may be detected ordetermined.

At this time, a fusion polypeptide in which a gankyrin polypeptide hasbeen fused to another peptide or polypeptide through gene engineeringtechnology may be used. Such another peptide or polypeptide that can besubjected to fusion include hemaglutinin (HA), FLAG, and the like, and afree gankyrin polypeptide can be detected or determined using antibodiesto another peptide or polypeptide that are subjected to fusion. Thus,the fusion polypeptides in which a gankyrin polypeptide and anotherpeptide or polypeptide have been fused through gene engineeringtechnology are useful in the present invention.

Samples expected to contain an agonist or an antagonist for use in thescreening method of the present invention include, for example,peptides, proteins, non-peptide compounds, synthetic compounds,microbial fermentation products, marine organism extracts, plantextracts, cell extracts, or animal cell extracts. These samples may benovel or known substances.

The screening method of the present invention is useful for detecting ordetermining an agonist or an antagonist having carcinogenicity.

It was found in the present invention that gankyrin polypeptidesinteract with Rb or NFκB. Since gankyrin polypeptides havetumorigenicity, agonists or antagonists of gankyrin polypeptides thatmodulate the binding of a gankyrin polypeptide and Rb or NFκB are usefulas pharmaceuticals.

Specifically, in order to conduct the above-mentioned screening method,the gankyrin polypeptide of the present invention or a sample expectedto contain the gankyrin polypeptide is first suspended in a buffersolution suitable for screening and then immobilized on a plate therebyto prepare a gankyrin polypeptide sample.

Any buffer solution may be used as long as it does not inhibit thebinding of a gankyrin polypeptide and, for example, a phasphate bufferof pH 6 to 8, Tris-HCl buffer, PBS, and HBSS may be used. In order toreduce non-specific binding, it is also possible to add protein such asbovine serum albumin, a surfactant such as CHAPS, Tween 80, digitonin,and the like. Furthermore, in order to suppress the decomposition of thegankyrin polypeptide with proteolyltic enzymes, inhibitors ofproteolytic enzymes such as PMSF, pepstatin, leupeptin, and the like canbe added.

Then, to the gankyrin polypeptide sample are added Rb or NFκB that hasbeen labeled with a radioisotope and an appropriate concentration ofsample, which are reacted at about 0 to 50° C. (preferably about 4 to37° C.) for about 0.5 to 24 hours (preferably about 0.5 to 3 hours).After the reaction, it is washed with an appropriate amount of bufferand the amount of radioactivity remaining in the gankyrin polypeptidesample is counted by a gamma counter or liquid scintillation counter. Inorder to determine non-specific binding at this time, anotherpolypeptide that does not interact with the gankyrin polypeptide issimilarly labeled and added to prepare a gankyrin polypeptide sample.The gankyrin polypeptide sample to which a buffer that does not containthe sample has been added is used as a negative control.

The amount of non-specific binding subtracted from the amount ofremaining radioactivity give the amount of specific binding. A samplethat reduces the specific binding as compared to the case when no samplewas added to the reaction can be selected as a candidate substance foran agonist or an antagonist of the gankyrin polypeptide.

An agonist or an antagonist of the gankyrin polypeptide obtained by thescreening method of the present invention can be applied to screening,for example, peptides, proteins, non-peptide compounds, syntheticcompounds, microbial fermentation products, marine organism extracts,plant extracts, cell extracts, or animal cell extracts using thescreening method. These samples may be novel or known substances.

An agonist or an antagonist of a gankyrin polypeptide is a substancethat inhibits the binding of the gankyrin polypeptide and Rb or NFκB.Substances obtained by addition, deletion or substitution of part of thestructure of an agonists or an antagonists of a gankyrin polypeptideobtained by the screening method of the present invention is alsoincluded into agonists or antagonists of gankyrin polypeptides obtainedby the screening method of the present invention.

When agonists or antagonists of gankyrin polypeptides obtained by thescreening method of the present invention are used as medicaments forhumans and mammals such as mice, rats, guinea pigs, rabbits, chickens,cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, theymay be used in a conventional method.

For example, they may be used, as desired, orally as sugar-coatedtablets, capsules, elixirs, and microcapsules, or parenterally in theform of sterile solutions with water or other pharmaceuticallyacceptable liquids or in the form of injections as suspensions. Forexample, agonists or antagonists of gankyrin polypeptides are producedin unit dosage forms required for generally accepted formulations bymixing with physiologically acceptable carriers, flavoring agents,excipients, vehicles, antiseptics, stabilizers, and binders. The amountof active ingredients in these formulations is designed to provide anindicated suitable range of doses.

Additives that can be blended into tablets and capsules include, forexample, binders such as gelatin, corn starch, tragacanth, gum Arabic,excipients such as crystalline cellulose, swelling agents such asalginate, lubricating agents such as magnesium stearate, sweeteningagents such as sucrose, lactose, and saccharin, and flavoring agentssuch as peppermint, Gaultheria adenothrix oil, or cherry. When theformulation unit form is a capsule, liquid carriers such as lipids canbe included to the above materials. Sterile compositions for injectioncan be formulated according to the conventional formulation method fordissolving or suspending active substances in a vehicle such asdistilled water for injection, and natural plant oils such as sesame oiland coconut oil.

As aqueous solutions for injection, there may be mentioned, for example,isotonic liquids such as physiological saline, glucose and otheradjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, and they may be used in combination with suitable solubilizingagents such as alcohols, specifically ethanol, polyalchohols including,for example, propylene glycol and polyethylene glycol, nonionicsurfactants such as polysorbate 80 (TM), and HCO-50.

An oily liquid includes, for example, sesame oil and soybean oil, whichmay be used together with solubilizing agents such as benzyl benzoateand benzyl alchohol. There may be also blended buffers such as phosphatebuffer and sodium acetate buffer, analgesics such as benzalkoniumchloride and procaine chloride, stabilizing agents such as benzylalchohol and phenol, and antioxidants. Prepared injections are usuallyfilled into suitable ampoules.

The dosage of agonists or antagonists of gankyrin polypeptides for ahuman adult (assuming the body weight of 60 kg) is, when given orally,usually about 0.1 to 100 mg/day, preferably about 1.0 to 50 mg/day, andmore preferably about 1.0 to 20 mg/day, though this may vary dependingon the medical conditions.

When given parenterally, the dose per administration for a human adult(assuming the body weight of 60 kg) of usually about 0.01 to 30 mg/day,preferably about 0.1 to 20 mg/day, and more preferably about 0.1 to 10mg/day in the case of injections is conveniently administered viaintravenous injection, though this may vary depending on the subject,subject organ, medical conditions, and the method of administration. Forother animals also, the amount converted in terms of the body weight of60 kg may be administered.

Anti-gankyrin polypeptide antibodies of the present invention can beobtained as monoclonal or polyclonal antibodies using known methods.

Monoclonal antibodies can be obtained by using a gankyrin polypeptide asa sensitizing antigen, which is immunized in a conventional method forimmunization, by fusing the immune cells thus obtained with known parentcells, and screening monoclonal antibody-producing cells using a knownscreening method.

Specifically, monoclonal or polyclonal antibodies may be generated asfollows.

For example, though the gankyrin polypeptide to be used as a sensitizingantigen for generation of antibodies is not limited by the animalspecies from which the antibodies are obtained, it is preferably derivedfrom a mammal such as humans, mice, or rats. These gankyrin polypeptidesderived from humans, mice, or rats can be obtained using the genesequences disclosed in the present invention.

According to the present invention, gankyrin polypeptides that have thebiological activity of all the gankyrin polypeptides disclosed in thepresent invention can be used as the gankyrin polypeptide for use as asensitizing antigen. As fragments of gankyrin polypeptides, there may bementioned, for example, C-terminal fragments of gankyrin polypeptides.As used herein “anti-gankyrin polypeptide antibody” means an antibodythat specifically reacts to the full-length or fragments of a gankyrinpolypeptide.

Genes encoding a gankyrin polypeptide or fragments thereof may beinserted to a known expression system to transform the host celldescribed herein, and the desired gankyrin or the fragments thereof areobtained by a known method from the inside or the outside of the hostcell and then the gankyrin polypeptide may be used as a sensitizingantigen. Alternatively, cells that express gankyrin polypeptide orlysates thereof may be used as a sensitizing antigen.

Mammals to be immunized with the sensitizing antigen are notspecifically limited, and they are preferably selected in considerationof their compatibility with the parent cell for use in cell fusion. Theygenerally include rodents, lagomorphs, and primates.

Rodents include, for example, mice, rats, hamsters, and the like.Lagomorphs include, for example, rabbits. Primates include, for example,monkeys. As monkeys, catarrhines (Old-World monkeys) such as cynomolgi,rhesus monkeys, sacred baboons, chimpanzees etc. are used.

Immunization of animals with a sensitizing antigen is carried out usinga known method. A general method, for example, involves theintraperitoneal or subcutaneous administration of a sensitizing antigento the mammal. Specifically, a sensitizing antigen which has beendiluted and suspended in an appropriate amount of phosphate bufferedsaline (PBS) or physiological saline etc. is mixed, as desired, with anappropriate amount of a common adjuvant, for example Freund's completeadjuvant. After being emulsified, it is preferably administered to themammal for several times every 4 to 21 days. Alternatively a suitablecarrier may be used at the time of immunization of the sensitizingantigen. After such immunization, the increase in the desired antibodylevels in the serum is confirmed by a conventional method.

In order to obtain polyclonal antibodies to a gankyrin polypeptide, theblood of the mammal that was sensitized with the antigen is removedafter the increase in the desired antibody levels in the serum has beenconfirmed. Serum is separated from the blood. As polyclonal antibodies,serum containing the polyclonal antibodies may be used, or, as desired,the fraction containing the polyclonal antibodies may be isolated fromthe serum.

In order to obtain monoclonal antibodies, immune cells of the mammalthat was sensitized with the antigen are removed and are subjected tocell fusion after the increase in the desired antibody levels in theserum has been confirmed. At this time preferred immune cells that aresubjected to cell fusion include, in particular, the spleen cells.

The mammalian myeloma cells as the other parent cells which aresubjected to cell fusion with the above-mentioned immune cellspreferably include various known cell lines such as P3 (P3X63Ag8.653)(Kearney, J. F. et al., J. Immunol. (1979) 123: 1548-1550), P3X63Ag8.U1(Yelton, D. E., et al., Current Topics in Microbiology and Immunology(1978) 81: 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol.(1976) 6: 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8:405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276: 269-270), FO (deSt. Groth, S. F. and Scheidegger, D., J. Immunol. Methods (1980) 35:1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148: 313-323), R210(Galfre, G. et al., Nature (1979) 277: 131-133) and the like.

Cell fusion between the above immune cells and the myeloma cells may beessentially conducted in accordance with a known method such as isdescribed in Milstein et al. (Kohler, G. and Milstein, C., MethodsEnzymol. (1981) 73: 3-46) and the like.

More specifically, the above cell fusion is carried out in theconventional nutrient broth in the presence of, for example, a cellfusion accelerator. As the cell fusion accelerator, for example,polyethylene glycol (PEG), Sendai virus (HVJ) and the like may be used,and, in addition, an adjuvant such as dimethyl sulfoxide etc. may beadded, as desired, to enhance the efficiency of the fusion.

The preferred ratio of the immune cells and the myeloma cells to be usedis, for example, 1 to 10 times more immune cells than the myeloma cells.Examples of culture media to be used for the above cell fusion includeRPMI1640 medium and MEM culture medium suitable for the growth of theabove myeloma cell lines, and the conventional culture medium used forthis type of cell culture and, besides, a serum supplement such as fetalcalf seran (FCS) may be added.

In cell fusion, predetermined amounts of the above immune cells and themyeloma cells are mixed well in the above culture liquid, to which a PEGsolution previously heated to about 37° C., for example a PEG solutionwith a mean molecular weight of about 1000 to 6000, is added at aconcentration of 30 to 60% (w/v), and mixed to obtain the desired fusioncells (hybridomas). Then, by repeating the sequential addition of asuitable culture liquid and centrifugation to remove the supernatant,cell fusion agents etc. which are undesirable for the growth of thehybridoma can be removed.

Said hybridoma is selected by culturing in a conventional selectionmedium, for example, the HAT culture medium (a culture liquid containinghypoxanthine, aminopterin, and thymidine). Culturing in said HAT culturemedium is continued generally for a period of time sufficient to effectkilling of the cells other than the desired hybridoma (non-fusioncells), generally several days to several weeks. Then, the conventionallimiting dilution method is conducted in which the hybridomas thatproduce the desired antibody are screened and cloned.

In addition to obtaining the above hybridoma by immunizing an animalother than the human with an antigen, it is also possible to sensitizehuman lymphocytes infected with EB virus with a gankyrin polypeptide,cells expressing a gankyrin polypeptide, or their lysates in vitro, andto allow the resulting sensitized lymphocytes to be fused with ahuman-derived myeloma cell having a permanent division potential, forexample U266, and thereby to obtain a hybridoma producing the desiredhuman antibody having the activity of binding the gankyrin polypeptide(see Japanese Unexamined Patent Publication (Kokai) No. 63(1988)-17688).

Furthermore, a transgenic animal having a repertoire of human antibodygenes is immunized with the gankyrin polypeptide, cells expressing thegankyrin polypeptide or lysates thereof to obtain the anti-gankyrinpolypeptide antibody-producing cells, which are used to obtain humanantibody against the gankyrin polypeptide using hybrodomas fused tomyeloma cells (see International Patent Publication WO 92-03918, WO93-2227, WO 94-02602, WO 94-25585, wO 96-33735 and WO 96-34096).

The monoclonal antibody-producing hybridomas thus constructed can besubcultured in the conventional culture liquid, or can be stored for aprolonged period of time in liquid nitrogen.

In order to obtain monoclonal antibodies from said hybridoma, there maybe employed a method in which said hybridoma is cultured in theconventional method and the antibodies are obtained as the culturesupernatant, or a method in which the hybridoma is administered to andgrown in a mammal compatible with said hybridoma and the antibodies areobtained as the ascites. The former method is suitable for obtaininghigh-purity antibodies, whereas the latter is suitable for a large scaleproduction of antibodies.

In addition to using a hybridoma to produce an antibody, immune cellsthat produce the desired antibody, for example the sensitizedlymphocytes that have been immortalized with an oncogene, may be used toobtain the antibody.

A monoclonal antibody thus produced can also be obtained as arecombinant antibody by recombinant gene technology. For example, ananti-gankyrin polypeptide antibody gene may be cloned from the hybridomaor an immune cell such as a sensitized lymphocyte that producesantibodies, and is integrated into a suitable vector which is thenintroduced into a host to produce a recombinant antibody. Recombinantantibodies may also be used in the present invention (see, for example,Borrebaeck, C. A. K., and Larrick, J. W., THERAPEUTIC MONOCLONALANTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD.1990).

Specifically, mRNA encoding the variable region (V region) ofanti-gankyrin polypeptide antibody can be isolated from a hybridoma thatproduces the anti-gankyrin polypeptide antibody. The isolation of mRNAis conducted by preparing total RNA using a known method such as theguanidine ultracentrifugation method (Chirgwin, J. M. et al.,Biochemistry (1979) 18, 5294-5299), the AGPC method (Chomzynski, P. andSacci, N., Anal. Biochem. (1987) 162, 156-159), and then purifying mRNAfrom the total RNA using the mRNA Purification Kit (Pharmacia) and thelike. Alternatively, mRNA can be prepared directly using the QuickPrepmRNA Purification kit (Pharmacia).

The mRNA obtained is used to synthesize the cDNA of the gene using areverse transcriptase. The synthesis of cDNA can be effected using theAMV Reverse Transcriptase First-strand cDNA Synthesis Kit (SeikagakuKogyo), and the like. Alternatively, for the synthesis and amplificationof cDNA, the 5′-Ampli FINDER RACE kit (CLONTECH) and the 5′-RACE method(Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17,2919-2932) that employs the polymerase chain reaction (PCR) may be used.

A DNA fragment of interest may be prepared from the PCR product thusobtained and ligated to a vector DNA. Furthermore, a recombinant vectoris constructed from this, which is then introduced into E. coli forselection of colonies to prepare the desired recombinant vector. Thebase sequence of the desired DNA may be confirmed by a known method suchas the dideoxy nucleotide chain termination method. Once the desired DNAencoding the V region of anti-gankyrin polypeptide antibody has beenobtained, it may be ligated to DNA encoding the constant region (Cregion) of the desired antibody, which is then integrated into anexpression vector. Alternatively, the DNA encoding the V region of theantibody may be integrated into an expression vector which alreadycontains DNA encoding the C region of the antibody. The C region ofantibody may be the one derived from the same animal species as the Vregion or the one derived from the different animal species from the Vregion.

In order to produce the anti-gankyrin polypeptide antibody for use inthe present invention, the antibody gene is integrated as describedbelow into an expression vector so as to be expressed under the controlof the expression regulatory region, for example an enhancer and/or apromoter. Subsequently, the expression vector may be transformed into ahost cell and the antibody can then be expressed therein.

For the expression of an antibody, DNA encoding the heavy chain (Hchain) or the light chain (L chain) of the antibody may be separatelyintegrated into an expression vector and the hosts are transformedsimultaneously, or DNA encoding the H chain and the L chain may beintegrated into a single expression vector and the host is transformedtherewith (see International Patent Application WO 94-11523).

Antibodies for use in the present invention may be antibody fragments ormodified versions thereof as long as they bind to gankyrin polypeptides.For example, as fragments of antibody, there may be mentioned Fab,F(ab′)2, Fv or single-chain Fv (scFv) in which Fv's of the H chain andthe L chain were ligated via a suitable linker. Specifically antibodiesare treated with an enzyme, for example, papain or pepsin, to produceantibody fragments, or genes encoding these antibody fragments areconstructed, and then introduced into an expression vector, which isexpressed in a suitable host cell (see, for example, Co, M. S. et al.,J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,Methods in Enzymology (1989) 178, 476-496; Plucktrun, A. and Skerra, A.,Methods in Enzymology (1989) 178, 476-496; Lamoyi, E., Methods inEnzymology (1986) 121, 652-663; Rousseaux, J. et al., Methods inEnzymology (1986) 121, 663-669; Bird, R. E. et al., Trends Biotechnol.(1991) 9, 132-137).

scFv can be obtained by ligating the V region of the H chain and the Vregion of the L chain of an antibody. In the scFv, the V region of the Hchain and the V region of the L chain are ligated via a linker,preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad.Sci. U.S.A. (1988) 85, 5879-5883). The V region of the H chain and the Vregion of the L chain in the scFv may be derived from any of theabove-mentioned antibodies. As the peptide linker for ligating the Vregions, any single-chain peptide comprising, for example, 12-19 aminoacid residues may be used.

DNA encoding scFv can be obtained using DNA encoding the H chain or theH chain V region of the above antibody and DNA encoding the L chain orthe L chain V region of the above antibody as the template by amplifyingthe portion of the DNA encoding the desired amino acid sequence amongthe above sequences by the PCR technique together with the primer pairspecifying the both ends thereof, and by further amplifying thecombination of DNA encoding the peptide linker portion and the primerpair which defines that both ends of said DNA are ligated to the H chainand the L chain, respectively.

Once DNAs encoding scFv are constructed, an expression vector containingthem and a host transformed with said expression vector can be obtainedby the conventional methods, and scFv can be obtained using theresultant host by the conventional methods.

These antibody fragments may be antibody fragments part of which haveundergone mutation, substitution, deletion, or insertion. These antibodyfragments can also be produced by obtaining the gene thereof in asimilar manner to that mentioned above and by allowing it to beexpressed in a host. “Antibody” as used in the claim of the presentapplication encompasses these antibody fragments.

As modified antibodies, anti-gankyrin polypeptide antibodies associatedwith various molecules such as polyethylene glycol (PEG) can be used.“Antibody” as used in the claim of the present application encompassesthese modified antibodies. These modified antibodies can be obtained bychemically modifying the antibodies thus obtained. These methods havealready been established in the art.

The anti-gankyrin polypeptide antibody of the present invention can beobtained as chimeric or humanized antibody using a known method.

The antibody gene constructed as above can be expressed by a knownmethod to obtain the antibody. For example, promoters/enhancers forproduction of the gankyrin polypeptide described herein can be used.

For the production of anti-gankyrin polypeptide antibody for use in thepresent invention, any production system can be used, and the productionsystem for the production of gankyrin polypeptide described herein canbe used. For example, the production system for anti-gankyrinpolypeptide antibody preparation comprises the in vitro or the in vivoproduction system. As the in vitro production system, there can bementioned a production system which employs eukaryotic cells and theproduction system which employs prokaryotic cells. As the in vitroproduction system, there can be mentioned methods that use animals orthat use plants. When animals are used, there are the production systemswhich employ mammals and insects.

As mammals, goats, pigs, sheep, mice, and cattle and, for example,transgenic animals thereof can be used (Glaser V., SPECTRUMBiotechnology Applications, 1993). Also as insects, silkworms can beused. Furthermore, when plants are used, tabacco, for example, such asNicotiana tabacum can be used (Ma, J. K. et al., Eur. J. Immunol. (1994)24, 131-138).

When an antibody is produced in in vitro or in vivo production systems,as described above, DNA encoding the H chain or the L chain of theantibody may be separately integrated into an expression vector and thehosts are transformed simultaneously, or DNA encoding the H chain andthe L chain may be integrated into a single expression vector and thehost is transformed therewith (see International Patent Application WO94-11523).

Antibodies produced and expressed as described above can be separatedfrom the inside or outside of the host cell and then may be purified tohomogeneity. Separation and purification of the antibody for use in thepresent invention may be accomplished by, but not limited to, theseparation and the purification methods conventionally used for proteinpurification.

For example, there can be mentioned chromatography columns such asaffinity chromatography, filtration, ultrafiltration, salting-out,dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusingand the like, from which methods can be selected and combined asappropriate for separation and purification of antibody (Antibodies: ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988).

As columns for use in affinity chromatography, there can be mentionedProtein A column and Protein G column. Examples of the carriers used inthe Protein A column are Hyper D, POROS, Sepharose F. F. (Pharmacia) andthe like.

As chromatography other than the above-mentioned affinitychromatography, there can be mentioned, for example, ion exchangechromatography, hydrophobic chromatography, gel-filtration, reversephase chromatography, adsorption chromatography, and the like(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual. Ed Daniel R. Marshak et al., Cold Spring HarborLaboratory Press, 1986). These chromatographies can be carried out usinga liquid chromatography such as HPLC, FPLC.

The concentration of antibody obtained as above can be determined by themeasurement of absorbance or by the enzyme-linked immunosorbent assay(ELISA) and the like. Thus, when absorbance measurement is employed, theantibody obtained is appropriately diluted with PBS and then theabsorbance is measured at 280 nm. In the case of human antibody,calculation is conducted using 1.40 OD at 1 mg/ml, though the absorptioncoefficient varies depending on the species and the subclass.

When the ELISA method is used, measurement is conducted as follows.Thus, 100 μl of goat anti-human IgG diluted to 1 μg/ml in 0.1 Mbicarbonate buffer, pH 9.6, is added to a 96-well plate (manufactured byNunc), and is incubated overnight at 4° C. to immobilize the antibody.After blocking, 100 μl each of appropriately diluted antibody of thepresent invention or a sample containing the antibody, or 100 μl ofhuman IgG as the concentration standard is added, and incubated at roomtemperature for 1 hour.

After washing, 100 μl of 5000-fold diluted alkaline phosphatase-labeledanti-human IgG is added, and incubated at room temperature for 1 hour.After washing, the substrate solution is added and incubated, followedby the measurement of absorbance at 405 nm using the MICROPLATE READERModel 3550 (Bio-Rad) to calculate the concentration of the desiredantibody.

Alternatively, BIAcore (Pharmacia) can be used for the measurement ofantibody concentration.

The activity of anti-gankyrin polypeptide antibody of the presentinvention can be evaluated by a known method. For example, the activityof anti-gankyrin polypeptide antibody of the present invention can beevaluated by adding ¹²⁵I-labeled anti-gankyrin polypeptide antibody to aplate on which gankyrin polypeptide has been immobilized, washing theplate according to a known method, and then measuring the radioactivity(Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor Laboratory, 1988).

As methods for determining the antigen-binding activity of anti-gankyrinpolypeptide antibody for use in the present invention, there can be usedELISA, EIA (enzymeimmunoassay), RIA (radioimmunoassay), or thefluorescent antibody method.

When ELISA is employed, for example, a gankyrin polypeptide is added toa plate onto which anti-gankyrin polypeptide antibody has beenimmobilized, and then samples containing the desired anti-gankyrinpolypeptide antibody, for example a culture supernatant of anti-gankyrinpolypeptide antibody-producing cells or purified antibody, are addedthereto. The second antibody that recognizes the anti-gankyrinpolypeptide antibody labeled with an enzyme such as alkaline phosphataseis added, and the plate is incubated, washed. Then the enzyme substrateis added, and the absorbance is measured to evaluate the antigen-bindingactivity. As the gankyrin polypeptide, a fragment of the gankyrinpolypeptide, a fragment comprising the C-terminal thereof, or a fragmentcomprising the N-terminal thereof may be used. For the evaluation of theactivity of the anti-gankyrin polypeptide antibody of the presentinvention, BIAcore (Pharmacia) can be used.

By using such methods, a method of detecting or determining a gankyrinpolypeptide may be conducted, said method comprising contacting saidantibody to a sample expected to contain said gankyrin polypeptide anddetecting or determining an immune complex between said antibody andsaid gankyrin polypeptide.

Specifically, when ELISA is employed, for example, a sample containing agankyrin polypeptide is added to a plate onto which anti-gankyrinpolypeptide antibody has been immobilized, and then anti-gankyrinpolypeptide antibody is added thereto.

The second antibody that recognizes the anti-gankyrin polypeptideantibody labeled with an enzyme such as alkaline phosphatase is added,and the plate is incubated and washed. Then, after adding the enzymesubstrate such as p-nitrophenyl phosphate thereto and determiningabsorbance, the presence of the gankyrin polypeptide in the sample canbe evaluated. As the gankyrin polypeptide, a fragment of the gankyrinpolypeptide, a fragment comprising the C-terminal thereof, or a fragmentcomprising the N-terminal thereof may be used. For the evaluation of theactivity of the anti-gankyrin polypeptide antibody of the presentinvention, BIAcore (Pharmacia) can be used.

The method of detecting or determining the gankyrin polypeptide of thepresent invention is important in various experiments that employgankyrin polypeptides since it can specifically detect or determinegankyrin polypeptides.

The present invention includes a nucleotide (DNA or RNA) capable ofselectively hybridizing the gene of the present invention or anucleotide derivative such as an antisense oligonucleotide or ribozymeand the like. The present invention also includes an antisenseoligonucleotide that hybridizes any of the sites in the nucleotidesequence as set forth in SEQ ID NO: 1. The antisense oligonucleotide ispreferably an antisense oligonucleotide to at least 20 or morecontiguous nucleotides in the base sequence as set forth in SEQ IDNO: 1. More preferably, it is an antisense oligonucleotide in which saidat least 20 or more contiguous nucleotides in the nucleotide sequence asset forth in SEQ ID NO: 1 contain a translation initiation codon. Forexample, the antisense oligonucleotide of the present invention containsSEQ ID NO: 8. Furthermore, for example, the antisense oligonucleotide ofthe present invention is one that contains SEQ ID NO: 9.

As used herein “antisense oligonucleotide” may contain one or aplurality of nucleotide mismatches as long as nucleotides correspondingto nucleotides constituting a given region of DNA or mRNA are allcompelementary and DNA or mRNA and the oligonucleotide can selectivelyand stably hybridize to the base sequence as set forth in SEQ ID NO: 1.“Selectively and stably hybridize” as used herein means that they have ahomology of at least 70%, preferably 80%, more preferably 90%, morepreferably 95% or more of the base sequence on at least 20, preferably30 contiguous nucleotide sequence regions.

According to one embodiment of the present invention, the antisenseoligonucleotide has the nucleotide sequence as set forth in SEQ ID NO:8. Furthermore, according to one embodiment of the present invention,the antisense oligonucleotide has the nucleotide sequence as set forthin SEQ ID NO: 9.

When the oligonucleotide derivative for use in the present invention isa deoxyribonucleotide, each structure is as shown in formula (I):

wherein X may be independently any of oxygen (O), sulfur (S), a loweralkyl group, or a primary amine or a secondary amine; Y may beindependently any of oxygen (O) or sulfur (S); B is selected from thegroup consisting of adenine, guanine, thymine, and cytosine, and ismainly a complementary oligonucleotide to the DNA or the mRNA of thehuman gankyrin gene; R is independently hydrogen (H) or adimethoxytrityl group or a lower alkyl group; and n is 7 to 28.

Preferred oligonucleotide derivatives may be not only unmodifiedoligonucleotides, but, as described hereinbelow, modifiedoligonucleotides. Examples of such modifications include, for example,lower alkyl phosphonate-modifications such as the above-mentionedmethylphosphonate type or the ethylphosphonate type, and thephosphorothioate modifications or the phosphoroamidate modifications.

Examples of

are represented by the following formula (II):

These antisense oligonucleotide derivatives can be obtained by aconventional method as shown below. An oligonucleotide of Formula (1) inwhich X and Y are O may be readily synthesized using a commerciallyavailable DNA synthesizer (for example the one manufactured by AppliedBiosystems). Synthesis can be effected by the solid phase synthesisusing hydrogen phosphonate (T. Atkinson, & M. Smith, in OligonucleotideSynthesis: A Practical Approach, ed. M. J. Gait, IRL Press, 35-81(1984); M. H. Caruthers, Science 230, 281 (1985); A. Kume, et al., J.Org. Chem., 49, 2139 (1984); B. C. Froehler, et al., Tetrahedron Lett.27, 469 (1986); P. J. Garegg, et al., ibid, 27, 4051 (1986); B. S.Sproat, et al., in Oligonucleotide Synthesis: A Practical Approach, ed.M. J. Gait, IRL Press, 83-115 (1984); S. L. Beaucage & M. H. Caruthers,Tetrahedron Lett., 22, 1859-1862 (1981); M. D. Matteucci and M. H.Caruthers, Tetrahedron Lett., 21, 719-722 (1980); M. D. Matteucci & M.H. Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981)

A triester phosphate modification in which X is a lower alkoxy group canbe obtained by, for example, a conventional method in which anoligonucleotide that has been obtained by chemical synthesis is treatedwith a solution of tosyl chloride in DMF/methanol/2,6-lutidiene (MoodyH. M. et al., Nucleic Acids Res., 17, 4769-4782 (1989)).

An alkyl phosphonate modification in which X is an alkyl group can beobtained by, for example, using phosphoamidite (M. A. Dorman, et al.,Tetrahedron Lett. 40, 95-102 (1984); K. L. Agarwal & F. Riftina, NucleicAcids Res., 6, 3009-3024 (1979)).

A triester phosphorothioate modification in which X is S can be obtainedby a solid phase synthesis using sulfur (C. A. Stein, et al., NucleicAcids Res., 16, 3209-3221 (1988)), or a solid phase synthesis usingtetraethyltiraum disulfide (H. Vu and B. L. Hirschbein, TetrahedronLett. 32, 3005-3008 (1991).

A phosphorodithioate modification in which both X and Y are S can beobtained by, for example, a solid phase synthesis in which a bisamiditeis converted to a thioamidite, on which is acted sulfur to yield saidmodification (W. K.-D. Brill, et al., J. Am. Chem. Soc., 111, 2321-2322(1989)).

A phosphoroamidate modification in which X is a primary amine or asecondary amine can be obtained by, for example, a solid phase synthesisin which hydrogen phosphonate is treated with a primary or secondaryamine (B. Froehler, et al., Nucleic Acids Res., 16, 4831-4839 (1988)).Alternatively the amidite may be oxidized with tert-butyl hydroperoxideto yield said modification (H. Ozaki, et al., Tetrahedron Lett., 30,5899-5902 (1989)).

Purification and the confirmation of purity can be carried out by highperformance liquid chromatography and polyacrylamide gelelectrophoresis. The confirmation of molecular weight can be carried outby Electrospray Ionization Mass Spectrometry or Fast AtomBombardment-Mass Spectrometry. The antisense oligonucleotide of thepresent invention may be obtained by any synthetic method or from anysource as long as it has a sequence that hybridizes to the base sequenceof DNA or mRNA encoding a human gankyrin polypeptide.

The antisense oligonucleotide derivative of the present invention actson the human gankyrin polypeptide-producing cells, as shown hereinbelowin Example 7, to bind to DNA or mRNA encoding the human gankyrinpolypeptide and thereby to inhibit its transcription or translation andpromote the decomposition of mRNA, resulting in the suppression of humangankyrin polypeptide expression. Eventually it exhibits an effect ofsuppressing the actions of human gankyrin polypeptide. The actions ofhuman gankyrin polypeptide suppressed by the antisense oligonucleotidederivative of the present invention includes, for example, thesuppression of the colony-forming ability in soft agar by the cellsdescribed in Example 7.

The antisense oligonucleotide derivative of the present invention can bemixed with an appropriate base to formulate an external preparation suchas a liniment, a cataplasm and the like.

It can also be mixed, as desired, with an excipient, an isotonic agent,a solubilizer, a stabilizer, an antiseptic, a soothing agent or the liketo formulate a tablet, powder, granules, a capsule, a liposome capsule,an injection, a solution, a nasal drop, and the like as well as alyophilized preparation. They can be prepared according to aconventional method.

The antisense oligonucleotide derivative of the present invention may beapplied to the patient by either directly administering to the affectedarea of the patient or administering into the blood vessel therebyallowing the substance to be delivered to the affected area.Furthermore, an antisense encapsulating material that enhances prolongedaction and membrane permeability may be used. There may be mentioned,for example, liposome, poly-L-lysine, lipid, cholesterol, Lipofectin® (a1:1 (w/w) liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)and dioleoyl phophotidylethanolamine (DOPE) in membrane filtered water,from Invitrogen Corp.) or derivatives thereof.

Preferably the dosage of the antisense oligonucleotide derivative of thepresent invention can be adjusted as appropriate depending on thecondition of the patient to employ a preferred amount. For example,preferred dosage is in the range of 0.1 to 100 mg/kg, preferably 0.1 to50 mg/kg.

The antisense oligonucleotide of the present invention is useful ininhibiting the expression of gankyrin polypeptide and thereby insuppressing the biological activity of gankyrin polypeptide. Aninhibitor of the expression of gankyrin polypeptide containing theantisense oligonucleotide of the present invention can suppress thebiological activity, i.e. carcinogenicity, of gankyrin, and therefore,is useful as a therapeutic agent for cancer or hypertrophic disorders.

EXAMPLES

The present invention is now explained in more detail with reference tothe following examples.

Example 1 cDNA Cloning by the Subtraction Method

Using the subtraction method (Nakayama, H. et al., Develop. GrowthDiffer. (1996) 38, 141-151), cDNAs of the genes that are specificallyexpressed in hepatic cancer were cloned.

From surgical specimens of a 55-year old male patient, tissues of stage3 hepatic cancer as defined in the code of handling primary hepaticcancer (compiled by the Japan Hepatic Cancer Study Group) and the normalliver tissue were removed. From each tissue, total RNA was extractedusing the TRIsol reagent (manufactured by GIBCO BRL). From the totalRNA, double stranded cDNA was synthesized with oligo-dT primers usingthe cDNA synthesis kit (manufactured by Pharmacia). After digesting thecDNA with a restriction enzyme RsaI, a linker adapter (Nakayama, H. etal., Develop. Growth Differ. (1996) 38, 141-151) was added thereto, andthese cDNAs were amplified by the PCR method using pimers (Nakayama, H.et al., Develop. Growth Differ. (1996) 38, 141-151). When the cDNA fromthe normal liver tissue was amplified by the PCR method, primers thatwere end-labeled with biotin were used.

An excessive amount of double stranded cDNA derived from the normalliver tissue was mixed with a small amount of double stranded cDNAderived from hepatic cancer, and the mixture was then heat-denatured tomake it single-stranded, followed by annealing to double strands. Mostof the cDNAs derived from hepatic cancer tissue that were also expressedin the normal liver tissue hybridize with cDNAs derived from the normalliver tissue and come to have biotin labels. However, molecules that arespecific for the hepatic cancer tissue form double strands there betweenand thereby do not come to have biotin labels. Accordingly, doublestranded cDNAs having biotin labels were eliminated and cDNA moleculesspecific for the hepatic cancer tissue were concentrated.

cDNA molecules that are specific for the hepatic cancer tissue wereamplified by the PCR method and were concentrated by repeating the sameprocedure by 5 times. This gave 250 bp of cDNA fragments specific forthe hepatic cancer tissue derived from the human hepatic cancer tissue.In order to isolate the full-length cDNA, a cDNA library was constructedfrom a human placenta, the mouse NIH/3T3 cell line, and a rat placentaby a conventional method and were ligated to the λZAPII phage vector(manufactured by Strategene). Using the above 250 bp human cDNAfragments as a probe, the above placenta cDNA libraries were screenedunder a highly stringent condition.

Thus, they were hybridized in a hybridization solution (5×SSPE, 50%formamide, 5×Denhardt's solution, 0.5% SDS, 100 μg/ml denatured DNA, 10%dextran sulfate) at 42° C., followed by washing under a condition of1×SSC, 1.0% SDS, 65° C. (Sambrook, J. et al., Molecular Cloning, ColdSpring Harbor Laboratory Press (1989)). As a result, 1542 bp of humancDNA containing 678 bp Of ORF was obtained.

The 678 bp of ORF in this cDNA was PCR-amplified, which was then used asa probe in a screening of cDNA libraries of the rat placenta and mouseNIH/3T3 cell lines under a less stringent condition. Thus, they werehybridized in a hybridization solution (5×SSPE, 50% formamide,5×Denhardt's solution, 0.5% SDS, 100^(˜).tg/ml denatured DNA, 10%dextran sulfate) at 37° C., follow^(˜)d by washing under a condition of1×SSC, 1.0% SDS, 37° C. (Sambrook, J. et al., Molecular Cloning, ColdSpring Harbor Laboratory Press (1989)).

As a result, cDNAs derived from the rat and the mouse were isolated. Thenucleotide sequence of these cDNAs were determined using a conventionalmethod, and the nucleotide sequence in turn was used to determine aminoacid sequence. The estimated amino acid sequences of human, rat, andmouse cDNAs are shown by a one-letter code and are compared, as shown inthe following Table 1. The polypeptides having these amino acidsequences were designated as gankyrin. The human gankyrin gene and themouse gankyrin gene had a 90% homology on the base sequence level and a93% homology on the amino acid sequence level. On the other hand, humangankyrin gene and the rat gankyrin gene had a 91% homology on the basesequence level and a 94% homology on the amino acid sequence level.

TABLE 1 HumanMEGCVSNLMVCNLAYSGKLEELKESILADKSLATRTDQDSRTALHWACSAGHTEIVEFLL MouseMEGCVSNIMICNLAYSGKLDELKERILADKSLATRTDQDSRTALHWACSAGHTEIVEFLL RatMEGCVSNLMVCNLAYNGKLDELKESILADKSLATRTDQDSRTALHWACSAGHTEIVEFLL HumanQLGVPVNDKDDAGWSPLHIAASAGRDEIVKALLGKGAQVNAVNQNGCTPLHYAASKNRHE MouseQLGVPVNDKDDAGWSPLHIAASAGRDEIVKALLVKGAHVNSVNQNGCTPLHYAASKNRHE RatQLGVPVNEKDDAGWSPLHIAASAGRDEIVKALLIKGAQVNAVNQNGCTALHYAASKNRHE HumanIAVMLLEGGANPDAKDHYEATAMHRAAAKGNLKMIHILLYYKASTNIQDTEGNTPLHLAC MouseISVMLLEGGANPDAKDHYDATAMHRAAAKGNLKMVHILLFYKASTNIQDTEGNTPLHLAC RatIAVMLLEGGANPDAKNHYDATAMHRAAAKGNLKMVHILLFYKASYNIQDTEGNTPLHLAC HumanDEERVEEAKLLVSQGASIYIENKEEKTPLQVAKGGLGLILKRMVEG (SEQ ID NO: 2) MouseDEERVEEAKFLVTQGASIYIENKEEKTPLQVAKGGLGLILKRLAESEEASM (SEQ ID NO: 3) RatDEERVEEAKLLVTQGASIYIENKEEKTPLQVAKGGLGLILKRIVESEEASM (SEQ ID NO: 5)

The nucleotide sequence of human gankyrin is shown in SEQ ID NO: 1 andthe amino acid sequence thereof is shown in SEQ ID NO: 2. The nucleotidesequence of mouse gankyrin is shown in SEQ ID NO: 3 and the amino acidsequence thereof is shown in SEQ ID NO: 4. The nucleotide sequence ofrat gankyrin is shown in SEQ ID NO: 5 and the amino acid sequencethereof is shown in SEQ ID NO: 6. In addition, it was estimated that inthe amino acid sequences of gankyrins, the region from amino acid Met atposition 1 to amino acid Leu at position 13 is a signal sequence.

The amino acid sequence of the human gankyrin polypeptide thus obtainedhad 5.5 ankyrin repeats (Lambert, S. et al., Proc. Natl. Acad. Sci.U.S.A. (1990) 87, 1730-1734). This is shown in the following Table 2.

TABLE 2 ANK consensus G TPLHLAAR GHVEVVKLLLD GADVNA TK SEQ ID NO: 10   A  I SQ NNLDIAEV  K   NPD  D       V  K   T M R   Q   SI   N                     E 1st repeat DSRTALHWACSAGHTEIVEFLLQLGVPVNDKDD SEQID NO: 11 2nd repeat AGWSPLHIAASAGRDEIVKALLGKGAQVNAVNQ SEQ ID NO: 12 3rdrepeat NGCTPLHYAASKNRHEIAVMLLEGGANPDAKDH SEQ ID NO: 13 4th repeatYEATAMHRAAAKGNLKMIHILLYYKASTNIQDT SEQ ID NO: 14 5th repeatEGNTPLHLACDEERVEEAKLLVSQGASIYIENK SEQ ID NO: 15 6th repeatEEKTPLQVAKGGLGLILKRMVEG SEQ ID NO: 16

In this table, the upper 3 lines represent ankyrin sequences, and thebottom 6 lines represent ankyrin repeats in the amino acid sequence ofthe gankyrin polypeptide of the present invention.

Furthermore, FIG. 15 shows the site and the number of ankyrin repeats invarious proteins.

In order to determine the position of the gankyrin gene on thechromosome, fluorescence in situ hybridization was conducted. Thus,lymphocytes isolated from human blood were cultured in an minimumessential medium (MEM) supplemented with 10% fetal bovine serum andphytohemagglutinin (PHA) at 37° C. for 68 to 72 hours. This lymphocyteculture was treated with 0.18 mg/ml BrdU (manufactured by Sigma) tosynchronize the cell cycle of the cell population. The cells of whichthe cell cycle were synchronized were washed three times with aserum-free medium. After the cell cycle arrest was removed, they werecultured again in a MEM containing 2.6 μg/ml thymidine (manufactured bySigma) at 37° C. for 6 hours. Cells were collected, subjected to astandard procedure comprising a hypotonic treatment, fixation, and airdrying to prepare slides of chromosome specimens.

Phage DNA (Sambrook, J. et al. Molecular Cloning, supra) having an about8.0 kb gankyrin gene insert was biotin-labeled by nick translation withdATP and biotin-16-dCTP at 15° C. for one hour using the BRL BioNicklabel kit according to the instructions (Herg et al., Proc. Natl. Acad.Sci. U.S.A. 89:9509-9513 (1992)).

Using this as a probe, fluorescence in situ hybridization (FISH) wascarried out (Herg et al., Proc. Natl. Acad. Sci. U.S.A. 89:9509-9531(1992); Herg et al., Chromosoma, 102; 325-332 (1993)).

Thus, a slide was first treated at 55° C. for one hour to attach thechromosome on the glass slides. After an RNase treatment, the slide wasdenatured with 70% formamide in 2×SSC at 70° C. for 2 minutes, and thendehydrated with ethanol. The probe was denatured in a hybridizationmixture containing 50% formamide, 10% dextran sulfate and human cotI DNAat 75° C. for 5 minutes. After incubation at 37° C. for 15 minutes tosuppress the repeat sequence, the probe was added to the above denaturedslide. After overnight hybridization, the slide was washed with 50%formamide in 2×SSC at 37° C., and then in 1×SSC at 60° C.

After biotin was detected with fluorescence-labeled FITC-bound avidin(manufactured by Vector Laboratories), the slide was stained with DAPI(manufactured by Sigma), a fluorescent reagent for staining DNA, togenerate a G/Q-band pattern on the chromosome. Use of this methodenables the generation of graded band patterns peculiar to thechromosome with a fluorescent reagent for staining DNA, and chromosomeassigning and chromosome mapping (location).

Using a cooled charge-coupled device (CCD) camera (manufactured byPhotometrics) that is a TV camera capable of detecting a very weaklight, 21 metaphase (mitotic period) images were photographed. By overlapping the FISH signal and the DAPI band-forming chromosome, the FISHmap data was assigned to the chromosome band (Hery et al., Methods inMolecular Biology: In situ hybridization protocols (K. H. A Choo. ed),p. 35-49 (1994), Human Press, Clifton, N.J.).

Under the condition used, hybridization efficiency for this probe wasabout 81%. Thus, out of 100 mitotic figures tested, 80 figures haveshown signals on a pair of chromosomes. Since DAPI band formation wasused in order to identify specific chromosomes, signals from the probewere assigned to the long arm of the X chromosome. Furthermore, detailedpositions was determined by putting together 10 photographs. The resultis shown in FIG. 1. Since no other loci were detected by the FISH underthe condition used, the probe T4-11 was assigned to the chromosome Xregion q21.3-q22.2.

The result (fluorescence staining) of the in situ hybridization obtainedis shown in FIG. 2.

Example 2 A Study on the Expression Level of a Gankyrin Gene in theTissue

In order to study the expression of a gankyrin gene, various tissues andcells were homogenized in the TRIzol reagent (manufactured by GIBCOBRL). Total RNA (20 μg) was denatured, and was separated byelectrophoresis in a 1.0% agarose gel containing 2.2 M formaldehyde.

The gel was blotted to the Hybond N+ nylon membrane (manufactured byAmersham), and was hybridized to [α-³²P]dCTP-labeled cDNA fragment (250bp of human gankyrin cDNA) in a rapid hybridization buffer (Rapi-hybbuffer, manufactured by Amersham). After hybridization, the filter waswashed under a stringent condition comprising the wash buffer containing0.1×SSC and 0.1% SDS at 65° C. for 30 minutes, and then was exposed to afilm at −80° C. The filter was cut into strips, which were hybridizedagain to a probe for 18S rRNA as an internal standard. The expressionlevel of RNA was evaluated by quantifying the autoradiogram by ascanning densitometer (manufactured by Ato).

The result of samples from the hepatic cancer tissue (T) and the hepaticnon-cancer tissue (N) is shown in FIG. 3. The lower part represents theresult of the internal standard and the upper part represents the resultdetected with the cDNA probe of human gankyrin. It was shown thatgankyrin mRNA is expressed in excessive amounts in the hepatic cancertissue alone.

The result (positive tests/total tests) for the human cancer tissuesother than the liver is shown below.

RCC (kidney cell carcinoma)  0/20 Testicular carcinoma 0/5 Ovarycarcinoma 0/5 Gastric cancer 4/4

As a result, gankyrin mRNA was expressed in excessive amounts in thetissue of gastric cancer among the cancer tissues tested.

The results for various cell lines, i.e. human cell line HepG2 (lane 1),Hela (lane 2), K562 (lane 3), NC65 (lane 4), NEC8 (lane 5), T24 (lane6), and IMR90 (lane 7) are shown in FIG. 4. The expression of gankyrinmRNA was observed in some cell lines.

The result for various normal tissues, i.e. liver (lane 1), spleen cells(lane 2), pancreas (lane 3), heart (lane 4), adrenal (lane 5), thyroid(lane 6), placenta (lane 7), ovary (lane 8), testis (lane 9), kidney(lane 10), and lung (lane 11) are shown in FIG. 5. As shown in FIG. 5,there was little expression of gankyrin mRNA in normal human tissues.

The above result confirmed the specific and high expression of gankyrinmRNA in the cancer tissue.

Example 3 Preparation of Antibody to Gankyrin Polypeptide andImmunohistochemical Analysis Thereof

For preparation of anti-gankyrin polypeptide antibody, a peptideMet-Glu-Gly-Cys-Val-Ser-Asn-Leu-Met-Val-Cys-Asn-Leu-Ala-Tyr (SEQ ID NO:7) corresponding to the C-terminal region of gankyrin was immunized. Thepeptide was linked to keyhole limpet hemocyanin, and then was immunizedto a rabbit. On day 14, 42, and 56 after the immunization, the rabbitwas immunized again to obtain antiserum.

The antiserum obtained was affinity purified with the above immobilizedpeptide to obtain polyclonal antibody (Fmoc Chemistry, Research GeveticInc.). The reactivity and/or specificity to the gankyrin polypeptide ofthe present invention were confirmed by the Western blot analysis. Thus,an expression plasmid for a gankyrin gene was added to a TNT expressionsystem (manufactured by Promega) to obtain a translation product(gankyrin polypeptide). At this time, [³⁵S]-methionine was added to thesystem to synthesize [³⁵S]-methionine-labeled human gankyrinpolypeptide. Non-labeled gankyrin polypeptide was also synthesized usingthe same condition.

[³⁵S]-methionine-labeled and non-labeled human gankyrin polypeptideswere subjected to polyacrylamide gel electrophoresis (the condition isdescribed below), and the migration pattern of the non-labeled substancewas analyzed by the Western blot method. Thus, the migration gel of thenon-labeled substance was transferred to an Immobilon transfer membrane(manufactured by Millipore), and was blocked in a Tris buffer containing5% bovine serum albumin (BSA). Then the above blotting membrane wasincubated with the polyclonal antibody diluted 1:2000 to 1:10000 in aTris buffer, 0.1% Tween 20, and BSA at 4° C. for 16 hours.

The blotting membrane was repeatedly washed in the Tris buffer and 0.1%Tween 20 and then was incubated with anti-rabbit immunoglobulin antibodylabeled with horseradish peroxidase as the 2nd antibody at roomtemperature for 1 hour. After washing, it was allowed to develop colorwith an electrochemiluminescence reagent (manufactured by Amersham).

On the other hand, for the migration gel for the labeled substance, themigration pattern of gankyrin polypeptide was confirmed byautoradiography. As a result, a Western blot band of the non-labeledsubstance was observed corresponding to the autoradiography band of thelabeled substance, indicating that the polyclonal antibody recognizesthe gankyrin gene product.

In a Western blot analysis conducted simultaneously using a TNTexpression system containing no template DNA and a luciferase cDNAexpression product, the present polyclonal antibody, no such specificbands were observed, confirming the specificity of the polyclonalantibody to gankyrin polypeptide. FIG. 8A shows a result on products ofan in vitro-translated labeled gankyrin gene. The template (−) (lane 1),positive control (luciferase cDNA corresponding to about 60 kDa) (lane2), and gankyrin (lane 3). The result of Western blot analysis usinganti-gankyrin polypeptide antibody on the in vitro-translated gankyringene products (non-labeled) is shown in FIG. 8B. Each lane representsthe template (−) (lane 1), positive control (luciferase cDNAcorresponding to about 60 kDa) (lane 2), and gankyrin (lane 3).

FIG. 6 shows a result of an experiment in which gankyrin polypeptides inthe lysates of the hepatic non-cancer tissue (N) and the hepatic cancertissue (T) of three patients with hepatic cancer were detected by aWestern blot method using anti-gankyrin polypeptide antibody that wasaffinity-purified as described above. Similarly the level of mRNA shownin Example 2, more gankyrin polypeptides were detected in the hepaticcancer tissue than in the hepatic non-cancer tissue.

FIG. 7 shows the result of an experiment in which gankyrin polypeptidesfrom the total cell lysates of human cell lines, i.e. HepG2 (ATCCcatalogue 1994 (American Type Culture Collection)) (lane 1), HeLa (ATCCcatalogue 1994 (American Type Culture Collection)) (lane 2), T24 (ATCCcatalogue 1994 (American Type Culture Collection)) (lane 3), NC65(Hoehn, W. and Schroeder, F. H., Invest. Urol. (1978) 16, 106) (lane 4),NEC8 (Human Science Research Resource bank, Cell/gene catalogue, 2ndedition, 1995) (lane 5), Jurkat (ATCC catalogue 1994 (American TypeCulture Collection)) (lane 6), 293 (ATCC catalogue 1994 (American TypeCulture Collection)) (lane 7) and COS-7 (ATCC catalogue 1994 (AmericanType Culture Collection)) (lane 8) were detected by a Western blotmethod using the anti-gankyrin polypeptide antibody that wasaffinity-purified as described above. As a result, the expression ofgankyrin polypeptide was confirmed in all cell lines.

Total cell extracts and tissue extracts were prepared from 3˜5×10⁶ cellslysated in a modified buffer for radioisotope-labeledimmunoprecipitation comprising 50 mM Tris-HCl, pH 7.4, 400 mM NaCl, 1%SDS, 1% Triton X-100, 1% deoxycholic acid, and 5 mM EDTA, and thensubjected to ultrasonic treatment to shear DNA. Immediately before use,a phosphatase inhibitor comprising 15 mM β-glycerophosphate, 2 mM sodiumpyrophosphate and 1 mM Na₃VO₄, and a protease inhibitor comprisingaprotinin, leupeptin and phenylmethyl sulfonyl fluoride were added toall extracts.

Samples were separated on a SDS-polyacrylamide gel electrophoresis(PAGE), and the gel used in the SDS-PAGE was transferred to an Immobilontransfer membrane (manufactured by Millipore), followed by blocking in aTris buffer containing 5% bovine serum albumin (BSA). Then the aboveblotting membrane was incubated together with the above primaryantiserum or antibody diluted to 1:2000 to 1:10000 in the Tris buffer,0.1% Tween 20, and BSA at 4° C. for 16 hours. The blotting membrane wasrepeatedly washed in a Tris buffer and 0.1% Tween 20. Then it wasincubated with anti-rabbit immunoglobulin antibody or anti-mouseimmunoglobulin antibody labeled with horseradish peroxidase as the 2ndantibody at room temperature for 1 hour. After washing, it was allowedto develop color with an electrochemiluminescence reagent (manufacturedby Amersham).

Example 4 Characterization of Gankyrin Polypeptide

A 678 bp cDNA encoding human gankyrin polypeptide (Example 1) wasligated to PMKIT-NEO mammal expression vector (Shinsaibo Kogaku JikkenProtocol (New Cell Engineering Experiment Protocol) P. 259, TheUniversity of Tokyo, the Institute of Medical Science, ed., Shujunsha)in a sense and antisense direction. The SRa promoter in this vector candirect the constitutive synthesis of RNA from the inserted DNA. ThePMKIT-NEO vector has a neomycin resistant gene suitable for selection oftransformants.

Thirty micrograms of the plasmid construct was transfected into NIH 3T3cells (Jainchill, J. F. et al., J. Virol. (1969) 4, 549-553) by thecalcium phosphate method. Forty eight hours after the transfection, G418was added to the culture medium to a concentration of 1000 μg/ml.Individual colonies were isolated and were propagated for furtheranalysis. Thus, 5 sense clones, 5 antisense clines, and 5 control cloneswere established, and these clones were characterized by in vitrogrowth, morphology, cell cycle, and tumorigenicity

The doubling time was determined from the growth curve. The cells werecultured in a 2-layer soft agar comprising a bottom layer (DMEM, 10%FCS, 0.6% agar) and an upper layer (DMEM, 10% FCS, 0.3% agar). A 35 mmsoft agar plate was inoculated with 5×10³ cells, incubated at 37° C. for4 to 5 weeks, and then the cells were counted. The mean colony count ofthe clonies comprising 15 or more cells was 25±2 for the control clone,and 123±1 for the sense clone. Accordingly, the enhancement in theability of colony formation by the cells that express gankyrinpolypeptide in soft agar was demonstrated.

Tumorigenicity in the NIH 3T3 cell line was tested by transferringsubcutaneously 1×10⁶ cells to 4-week old female nude mice (Flanagan, S.P. Genet. Res. (1966) 8, 295-309). Each of 5 clonal cell linescomprising a mock construct and a clonal cell line containing a sensehuman gankyrin construct was subcutaneously transplanted to 3 mice (atotal of 18 mice) at 1×10⁶ cells/mouse. Tumor formation was observed for3 months after subcutaneous transplantation. Measurement of tumor wasconducted using a linear caliper in two right angles by the sameobserver. For cell cycle analysis, flow cytometry was used.

As a result, tumor formation when the control vector-containing cellswere inoculated was 0 clone (no tumor formation) out of 4 clones whereastumor formation when the sense vector-containing cell clones wereinoculated was 3 clones out of 4 clones. It is evident, therefore, thatthe transplantation of gankyrin polypeptide-expressing cells to miceshows tumorigenicity. In one clone in which no tumor was formed, theexpression level of gankyrin mRNA was lower than other 3 clones.

In a cultured human kidney cell line 293, apoptosis is induced byremoving serum from the culture medium and dead cells increase. Usingthis cell line, the effect of gankyrin gene on the induction ofapoptosis was investigated. Prior to the experiment, each gene wastransfected into the 293 cells by the calcium phosphate coprecipitationmethod using 10 μg of the above pMKIT-NEO vector, and a plurality ofclones in which each gene was stably introduced were obtained, whichwere used for the subsequent experiment.

It was shown, at this time, that the mean value of the colony focusnumber obtained from the G418 selection medium in three transfectionexperiments was 56±4 for the control clone, 70±4 for the sense clone,and 23±3 for the antisense clone. It was demonstrated, therefore, thatgankyrin polypeptide is involved in the promotion of cell growth andsuppression of apoptosis induction.

Using each 293 cell clone, 2×10⁵ cells were plated to a 60 mm tissueculture plate (Nunc GmbH). After the removal of serum, cells weretrypsinized and the numbers of the suspended cells and the attachedcells, and the number of dead cells were counted by trypan blue stainingto determine the ratio of the number of the dead cells to the number oftotal cells.

To analyze the number of apoptosis cells, a histochemical method wasemployed. Thus, cells of each clone were grown on a cover slip for 48hours until they reach a density of 60% saturation. After the removal ofserum, apoptosis cells were stained by the ApopTa apoptosis detectionkit (manufactured by Oncor), and examined under a light microscope tocount the ratio of the number of the apoptosis cells to that of totalcells.

In the above two experiments, the ratio (%) of the number of total cellsstained with trypan blue to the number of the total cells and the ratio(%) of the apoptosis cells to the number of total cells were 33±5% and45±5% for the control clone, and 59±6% and 30±2% for the sense clone,respectively. It was demonstrated, therefore, that apoptosis and celldeath by gankyrin gene products are suppressed.

For each clone, 2×10⁵ cells were plated to a 10 mm tissue culture plate.After incubation, serum was removed to induce apoptosis. In order toanalyze the fragmentation of gene DNA between nucleosomes, which ischaracteristic to the apoptosis cells, the above cells were scraped atpredetermined times and the supernatants were collected together withthe attached cells. The cells were resuspended in 0.25 ml of TBE (45 mMTris-borate, 1 mM EDTA, pH 8.0) containing 0.25% NP-40 and 0.1 mg/ml ofRNase.

After incubating at 37° C. for 30 minutes, the extracts were furthertreated with 1 mg/ml of proteinase K at 37° C. for 30 minutes. Then, 30μl of the extract was subjected to a 1.7% agarose gel electrophoresis inthe presence of 0.5 μg/ml of ethidium bromide. As a result, theladder-like electrophoretic pattern due to DNA fragmentation betweennucleosomes was decreased in the sense gankyrin gene-introduced cells ascompared to the control cells. It was demonstrated from these threeexperiments, therefore, that gankyrin gene products acts on apoptosisinduction in a suppressive manner. This is a characteristics observedfor many other tumorigenic genes, thereby indicating that the gankyringene is a tumorigenic gene.

Example 5 Interaction of Gankyrin Polypeptides

The human gankyrin cDNA obtained in Example 1 was ligated to apCMV4-3HA′ vector (Brockman, J. A. et al., Molecular and CellularBiology (1995) 15, 2809-2818) that has the cytomegalovirusenhancer/promoter and having the nucleotide sequence of influenza virushemagglutinin (HA) epitope to construct a plasmid pCMV4-3HA+gankyrinthat expresses a fusion polypeptide comprising gankyrin and influenzavirus HA.

Furthermore, a human gankyrin coding sequence was inserted to a vectorpGEX (manufactured by Pharmacia) to construct a plasmid that expresses afusion polypeptide comprising glutathione S-transferase (GST) andgankyrin.

Using 10 μg of pCMV4-3HA+gankyrin, the 293 cells were transientlytransfected by the calcium phosphate method. pGEX-gankyrin wasintroduced into E. coli, and the production of GST-gankyrin fusionpolypeptide was induced with 1 mM IPTG. After collecting the cells bycentrifuging at 4° C., the cells were dissolved by sonication in PBScontaining Triton X100. The cell lysate was mixed with the total cellextract from the 293 cell transformant, which was incubated at 4° C. for16 hours.

The GST-fused polypeptide was collected on glutathione-Sepharose 4B(manufactured by Pharmacia) and was analyzed by the Western blot methodusing anti-Rb antibody, anti-NFκB p50 antibody, and anti-NFκB p65 (allmanufactured by Santa Crutz).

The preparation of cell extracts and immunoprecipitates was conducted asfollows. To an IP buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl,2.5 mM EGTA, 1 mM DTT, 0.1% Tween 20, 10% glycerol, a proteaseinhibitor, and a phosphatase inhibitor, the cells were suspended,sonicated, and then centrifuged at 10000×g, 4° C., for 10 minutes. UsingProtein A-Sepharose CL4b (manufactured by Pharmacia) precoated withanti-HA antibody, anti-Rb antibody, anti-NFκB p50 antibody or anti-NFκBp65 antibody, the supernatant was precipitated at 4° C. for 16 hours.

Proteins that precipitated on the beads were washed ten times in the IPbuffer. The precipitate in the 2×SDS sample buffer was separated bySDS-polyacrylamide gel electrophoresis, and was analyzed by Western blotmethod with anti-HA antibody, anti-Rb antibody, anti-NFκB p50 antibodyor anti-NFκB p65 antibody.

The result is shown below.

FIG. 9 shows the result in which cell lysates were used in vitro. GSTalone or a fusion polypeptide of GST and gankyrin was expressed in E.coli, which was then harvested. The GST alone or the GST-gankyrin fusionpolypeptide was mixed with human 293 cells, and then precipitated withglutathione-bound Sepharose, which was electrophoresed.

The result is shown that was obtained after the electrophoresed gel wastransferred to a nitrocellulose membrane and was detected with (A)anti-Rb antibody, (B) anti-p50 antibody, and (C) anti-p65 antibody. In(A), lanes indicate, from left to right, the results of a human 293 celllysate alone, a precipitate obtained by mixing a human 293 cell lysatewith the above E. coli-expressed GST polypeptide and then byprecipitating it with glutathione-bound Sepharose, and a precipitateobtained by mixing a human 293 cell lysate with an E. coli-expressedfusion polypeptide and then by precipitating it with glutathione-boundSepharose.

In (B), lanes indicate, from left to right, the results of a human 293cell lysate alone, a precipitate obtained by mixing a human 293 celllysate with an E. coli-expressed GST polypeptide and then byprecipitating it with glutathione-bound Sepharose, and a precipitateobtained by mixing a human 293 cell lysate with an E. coli-expressedfusion polypeptide and then by precipitating it with glutathione-boundSepharose. In (C), lanes indicate, from left to right, the results of ahuman 293 cell lysate alone, a precipitate obtained by mixing a human293 cell lysate with an E. coli-expressed GST polypeptide and then byprecipitating it with glutathione-bound Sepharose, and a precipitateobtained by mixing a human 293 cell lysate with an E. coli-expressedfusion polypeptide and then by precipitating it with glutathione-boundSepharose.

The results of in vivo experiments on the cells are shown in FIG. 10. Byimmunoprecipitating the lysate of human 293 cells that express gankyrinfused to HA with (A) anti-Rb antibody, (B) anti-p50 antibody, or (C)anti-p65 antibody, and then by detecting with anti-HA antibody, anHA-fused gankyrin polypeptide was detected. In (A), lanes indicate theresults of a human 293 cell lysate that was transformed with a vectorcontaining no gankyrin gene and precipitated with a non-specificimmunoglobulin (lane 1) or anti-Rb antibody (lane 2), which was thenelectrophoresed and detected with anti-HA antibody, and a human 293 celllysate that was transformed with a vector containing a gankyrin gene andprecipitated with a non-specific immunoglobulin (lane 3) or anti-Rbantibody (lane 4), which was then electrophoresed and detected withanti-HA antibody.

In (B), lanes indicate the results of a human 293 cell lysate that wastransformed with a vector containing no gankyrin gene and precipitatedwith a non-specific immunoglobulin (lane 1) or anti-p50 antibody (lane2), which was then electrophoresed and detected with anti-HA antibody, ahuman 293 cell lysate that was transformed with a vector containing agankyrin gene and precipitated with a non-specific immunoglobulin (lane3) or anti-p50 antibody (lane 4), which was then electrophoresed anddetected with anti-HA antibody, and a human 293 cell lysate that wastransformed with a vector containing a gankyrin gene and thenelectrophoresed (lane 5) and detected with anti-HA antibody.

In (C), lanes indicate the results of a human 293 cell lysate that wastransformed with a vector containing no gankyrin gene and precipitatedwith a non-specific immunoglobulin (lane 1) or anti-p65 antibody (lane2), which was then electrophoresed and detected with anti-HA antibody, ahuman 293 cell lysate that was transformed with a vector containing agankyrin gene and precipitated with a non-specific immunoglobulin (lane3) or anti-p65 antibody (lane 4), which was then electrophoresed anddetected with anti-HA antibody, and a human 293 cell lysate that wastransformed with a vector containing a gankyrin gene and thenelectrophoresed (lane 5) and detected with anti-HA antibody.

The results of in vivo experiments on the cells are shown in FIG. 11. Byimmunoprecipitating the lysate of human 293 cells that express a fusionpolypeptide comprising HA and gankyrin, and then detecting with (A)anti-Rb antibody, or (B) anti-p50 antibody, Rb and p65 were detected,respectively.

In (A), lanes indicate the results of a human 293 cell lysate that wastransformed with a vector containing no gankyrin gene, electrophoresed,and detected with anti-Rb antibody (lane 1), a human 293 cell lysatethat was transformed with a vector containing no gankyrin gene andprecipitated with a non-specific immunoglobulin (lane 2) or anti-HAantibody (lane 3), which was then electrophoresed and detected withanti-Rb antibody, a human 293 cell lysate that was transformed with avector containing a gankyrin gene and precipitated with a non-specificimmunoglobulin (lane 3) or anti-HA antibody (lane 4), which was thenelectrophoresed and detected with anti-Rb antibody.

In (B), lanes indicate the results of a human 293 cell lysate that wastransformed with a vector containing no gankyrin gene and precipitatedwith a non-specific immunoglobulin (lane 1) or anti-HA antibody (lane2), which was then electrophoresed and detected with anti-p65 antibody,a human 293 cell lysate that was transformed with a vector containing agankyrin gene and precipitated with a non-specific immunoglobulin (lane3) or anti-HA antibody (lane 4), which was then electrophoresed anddetected with anti-p65 antibody, and a human 293 cell lysate that wastransformed with a vector containing a gankyrin gene and thenelectrophoresed (lane 5) and detected with anti-p65 antibody.

These results indicated that the gankyrin polypeptide interacts with Fbor NFκB in the cells (in vivo).

Example 6 Cell Cycle and Gankyrin Gene Expression

The cell cycle of NIH/3T3 cells was fixed at the early G1 period byserum starvation for 72 hours, and serum was added again to synchronizecell cycle. Cells were lyzed, mRNA was extracted, and detected usinggankyrin cDNA as a probe. Thus, after mRNA was amplified by the PCRmethod with cDNA of the coding region of mouse gankyrin as a template,mRNA was detected by the Northern blot method using ³²P-random primedlabeled product as a probe.

Flow cytometry was used for the analysis of cell cycle by various cellmeans. Thus, cells were washed in PBS containing no Ca²⁺ or Mg²⁺, andwere subjected to trypsin treatment. Then after the cells were washedwith DMEM containing 10% FCS and collected, the cells were washed againin the sample buffer and resuspended, and then were fixed in 70%ethanol. The cells were stained with PI (Propiodium iodine), anddetermined by a flow cytometer.

FIG. 12 shows the results. In FIG. 12, each lane represents the resultat 1 hour (lane 1), 3 hours (lane 2), 6 hours (lane 3), 9 hours (lane4), 12 hours (lane 5), 15 hours (lane 6), 18 hours (lane 7), 21 hours(lane 8), 24 hours (lane 9), 27 hours (lane 10), 30 hours (lane 11), and33 hours (lane 12) after the re-addition of serum. One to 9 hours (lane1 to 4) corresponds to the G1 period, 12 to 18 hours (lane 5 to 7)corresponds to the S period, 21 to 24 hours (lane 8 to 9) corresponds tothe G2+M period, and 27 hours or after (lane 10) returns to the G1period, again.

The result of the detection of mRNA expression in the cells grown atvarious concentrations is shown in FIG. 13. In this figure, the resultsof cell concentration 1×10⁶ cells/100 mm dish (lane 1), 2×10⁶ cells(lane 2), 3×10⁶ cells (lane 3), and 4×10⁶ cells (lane 4) are shown.

The result of detection of mRNA that is expressed during the process ofliver regeneration after partial hepatic resection in mice is shown inFIG. 14. The results before partial hepatic resection (lane 1), and 1hour (lane 2), 6 hours (lane 3), 24 hours (lane 4), 48 hours (lane 5),72 hours (lane 6), and 168 hours (lane 7) after partial hepaticresection are shown.

When gankyrin mRNA was examined by the NIH3T3 cells for which cell cyclewas synchronized, gankyrin expression was found to vary depending on thecell cycle. In the partially removed liver also, expression was found tovary depending on the cell cycle. These facts indicated that theexpression of gankyrin, associated with the progress of cell cycle,increases from the G1 phase to the S phase, suggesting its associationwith cell cycle regulation.

Example 7 Inhibitory Effects of an Antisense Strand on Hepatic CancerCells

Using a human hepatic cancer cell line HepG2, human gankyrin antisenseoligonucleotide derivative (sequence: ACCCCTCCATTTCGCTGTCC) (SEQ ID NO:8) and (TTAGACACACACCCCTCCAT) (SEQ ID NO: 9) were studied for theireffect of suppressing the growth of hepatic cells. As the culturemedium, RPMI1640 (Nissui) supplemented with 2% fetal calf serum (FCS)was used.

A 35 mm culture plate containing 1 ml culture liquid was incubatedovernight at 37° C. in a CO₂ incubator, to which were added 1×10² HepG2cells that were made single cells by a 0.25% trypsin treatment andpipetting. After 24 hours, it was replaced with 1 ml of the cultureliquied containing human gankyrin antisense oligonucleotide (0, 2.5, and10 μg/ml) that contains the initiation codon of a gankyrin gene, and wasfurther cultured for 4 days. A mass of 30 cells or more was defined as acolony, and the number of colonies was counted under an invertedmicroscope. Thus, the number of colonies when a sense oligonucleotidewas added was 95±7%, whereas the number of colonies when an antisenseoligonucleotide was added decreased to 70±5%, relative to 100% of thecontrol to which distilled water was added.

Human gankyrin antisense oligonucleotide had a suppressive effect on theformation of colonies. As a result, it was revealed that an antisenseoligonucleotide containing the initiation codon of gankyrin genesuppresses the growth of the hepatic cancer cell line HepG2 cells.

INDUSTRIAL APPLICABILITY

Since the gankyrin polypeptide of the present invention shows theelevation of colony-forming ability of cells, the suppression oftumorigenicity, and apoptosis induction in mice, it was shown to havecarcinogenecity. The gankyrin polypeptide and DNA encoding it are usefulfor elucidation of the mechanism of action of oncogenesis. A screeningmethod using a gankyrin polypeptide, antibody to a gankyrin polypeptide,a method of detecting or determininng a gankyrin polypeptide using it,and an antisense oligonucleotide to DNA encoding a gankyrin polypeptideare also useful for elucidation of the mechanism of action ofoncogenesis.

Reference to the microorganisms deposited under the Patent CooperationTreaty, Rule 13-2, and the name of the Depository organ

Depository Organ

-   Name: the National Institute of Bioscience and Human Technology,    Agency of Industrial Science and Technology-   Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan    Organism (1)-   Name: Escherichia coli DH5α [pBS-t4-11]-   Accession number: FERM BP-6128-   Date deposited: Sep. 29, 1997

1. A method for inhibiting the growth of hepatic cancer cells, whereinsaid hepatic cancer cells express (a) a polypeptide having the sequenceof amino acid 14 (Ala) to amino acid 226 (Gly) of SEQ ID NO: 2, or (b) apolypeptide encoded by the DNA sequence of SEQ ID NO: 1; the methodcomprising administering an antisense oligonucleotide capable ofspecifically and stably hybridizing to at least 20 contiguousnucleotides in the nucleotide sequence as shown in SEQ ID NO: 1, whereinthe antisense oligonucleotide is represented by the following formula:

wherein X is independently any of oxygen (O), sulfur (S), a lower alkylgroup, a primary amine or a secondary amine; Y is independently any ofoxygen (O) or sulfur (S); B is selected from the group consisting ofadenine, guanine, thymine and cytosine; R is independently hydrogen (H),a dimethoxytrityl group or a lower alkyl group; and n is 18 to 28,wherein the antisense oligonucleotide comprises the nucleotide sequenceas set forth as SEQ ID NO:
 8. 2. A method for inhibiting the growth ofhepatic cancer cells, wherein said hepatic cancer cells express (a) apolypeptide having the sequence of amino acid 14 (Ala) to amino acid 226(Gly) of SEQ ID NO: 2, or (b) a polypeptide encoded by the DNA sequenceof SEQ ID NO: 1; the method comprising administering an antisenseoligonucleotide capable of specifically and stably hybridizing to atleast 20 contiguous nucleotides in the nucleotide sequence as shown inSEQ ID NO: 1, wherein the antisense oligonucleotide is represented bythe following formula:

wherein X is independently any of oxygen (O), sulfur (S), a lower alkylgroup, a primary amine or a secondary amine; Y is independently any ofoxygen (O) or sulfur (S); B is selected from the group consisting ofadenine, guanine, thymine and cytosine; R is independently hydrogen (H),a dimethoxytrityl group or a lower alkyl group; and n is 18 to 28,wherein the antisense oligonucleotide comprises the nucleotide sequenceas set forth as SEQ ID NO:
 9. 3. The method of claim 1, wherein theantisense oligonucleotide is administered with an antisenseencapsulating material.
 4. The method of claim 2, wherein the antisenseoligonucleotide is administered with an antisense encapsulatingmaterial.
 5. The method of claim 3, wherein the antisense encapsulatingmaterial is a liposome, poly-L-lysine, lipid, cholesterol or a 1:1 (w/w)liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)and dioleoyl phophotidylethanolamine (DOPE).
 6. The method of claim 4,wherein the antisense encapsulating material is a liposome,poly-L-lysine, lipid, cholesterol a 1:1 (w/w) liposome formulation ofthe cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE).